COMPOSITIONS AND METHODS FOR MODULATION OF PROTEIN AGGREGATION

Abstract

In certain embodiments, the present disclosure provides methods comprising contacting a cell with a compound comprising a modified oligonucleotide complementary to a nucleic acid transcript. In certain such embodiments, the modified oligonucleotide does not interact or interacts poorly with a mRNP complex or granule. In certain such embodiments the modifications and/or motifs of the modified oligonucleotide do not promote interaction with a mRNP complex or granule. In certain embodiments, the present disclosure provides methods comprising contacting a cell with a compound comprising a modified oligonucleotide thereby reducing the size or amount of protein aggregation in the cell. In certain such embodiments, the protein aggregate is a mRNP granule. In certain such embodiments, the modifications and/or motifs of the modified oligonucleotide promote interaction with a protein aggregate, such as a mRNP granule, that results in disruption of the protein aggregate.

Description

SEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled CORE0144WOSEQ_ST25.txt, created Aug. 2, 2017, which is 144 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Protein aggregates, such as mRNP granules, are present in cells of patients with ALS, Parkinsons's Disease, and some forms of dementia, as well as other diseases. (See, e.g., Li et al. J Cell Biol, 201, 361-372 (2013); Seyfried et al. J Proteome Res, 11, 2721-2738 (2012); Ramaswami et al. Cell, 154, 727-736 (2013); Aulas and Vande Velde. Front Cell Neurosci, 9, 423 (2015); Shelkovnikova et al. Hum Mol Genet, 23, 5211-5226 (20140); and King et al. Brain Res, 1462, 61-80 (2012).) FUS/TLS (Fused in Sarcoma/Translocated in Sarcoma) and PSF/SFPQ (Polypyrimidine-Tract Binding Protein-Associated Splicing Factor/Splicing Factor Proline/Glutamine Rich) are ubiquitously expressed RNA-binding proteins with multifunctional roles in RNA metabolism. Both proteins contain prion-like, low complexity domains (LCD) that can facilitate aggregation. (See, e.g., Maziuk et al. Front Mol Neurosci 10, (2017); Xiang et al. Cell, 163, 829-839 (2015).)

[0003] Wild type FUS contains a PY-nuclear localization sequence (NLS). Some FUS mutants that disrupt the NLS lead to cytoplasmic accumulation and aggregation of FUS into cytoplasmic granules. (See, e.g., Shang and Huang. Brain Res, 1647, 65-78 (2016); Dormann et al. EMBO J, 31, 4258-4275 (2012); Zhang and Chook Proc Natl Acad Sci USA, 109, 12017-12021 (2012); Shelkovnikova et al. J Biol Chem, 288, 25266-25274 (2013).) In vitro, cytoplasmic FUS granules can be formed from the expression of a FUS mutant having a P525L mutation that is naturally occurring in some ALS patients.

SUMMARY OF THE INVENTION

[0004] Modified oligonucleotides can interact with proteins, including mRNP complexes or granules and/or proteins associated with mRNP complexes or granules. Such interactions may not be beneficial when the mRNP complex or granule sequesters the modified oligonucleotide in the cytoplasm, and the target of the modified oligonucleotide is located in the nucleus. Such interactions may be beneficial when aggregation of a mRNP granule is modulated, e.g., disrupted by the modified oligonucleotide. In certain embodiments, the present disclosure provides methods comprising contacting a cell with a compound comprising a modified oligonucleotide complementary to a nucleic acid transcript. In certain such embodiments, the modified oligonucleotide does not interact or interacts poorly with a mRNP complex or granule. In certain such embodiments the modifications and/or motifs of the modified oligonucleotide do not promote interaction with a mRNP complex or granule. In certain embodiments, the present disclosure provides methods comprising contacting a cell with a compound comprising a modified oligonucleotide thereby reducing the size or amount of protein aggregation in the cell. In certain such embodiments, the protein aggregate is a mRNP granule. In certain such embodiments, the modifications and/or motifs of the modified oligonucleotide promote interaction with a protein aggregate, such as a mRNP granule, that results in disruption of the protein aggregate.

DETAILED DESCRIPTION OF THE INVENTION

[0005] Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms, such as "includes" and "included", is not limiting.

[0006] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Definitions

[0007] As used herein, "2'-deoxynucleoside" means a nucleoside comprising 2'-H(H) ribosyl sugar moiety, as found in naturally occurring deoxyribonucleic acids (DNA). In certain embodiments, a 2'-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil).

[0008] As used herein, "2'-fluoro" or "2'-F" means a 2'-F in place of the 2'-OH group of a ribosyl ring of a sugar moiety.

[0009] As used herein, "2'-substituted nucleoside" or "2-modified nucleoside" means a nucleoside comprising a 2'-substituted or 2'-modified sugar moiety. As used herein, "2'-substituted" or "2-modified" in reference to a sugar moiety means a sugar moiety comprising at least one 2'-substituent group other than H or OH.

[0010] As used herein, "ALS" means amyotrophic lateral sclerosis.

[0011] As used herein, "antisense activity" means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.

[0012] As used herein, "antisense compound" means a compound comprising an antisense oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group.

[0013] As used herein, "antisense oligonucleotide" means an oligonucleotide having a nucleobase sequence that is at least partially complementary to a target nucleic acid.

[0014] As used herein, "ameliorate" in reference to a method means improvement in at least one symptom and/or measurable outcome relative to the same symptom or measurable outcome in the absence of or prior to performing the method. In certain embodiments, amelioration is the reduction in the severity or frequency of a symptom or the delayed onset or slowing of progression in the severity or frequency of a symptom and/or disease.

[0015] As used herein, "bicyclic nucleoside" or "BNA" means a nucleoside comprising a bicyclic sugar moiety. As used herein, "bicyclic sugar" or "bicyclic sugar moiety" means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the bicyclic sugar moiety does not comprise a furanosyl moiety.

[0016] As used herein, "cEt" or "constrained ethyl" means a ribosyl bicyclic sugar moiety wherein the second ring of the bicyclic sugar is formed via a bridge connecting the 4'-carbon and the 2'-carbon, wherein the bridge has the formula 4'-CH(CH.sub.3)--O-2', and wherein the methyl group of the bridge is in the S configuration.

[0017] As used herein, "cleavable moiety" means a bond or group of atoms that is cleaved under physiological conditions, for example, inside a cell, an animal, or a human.

[0018] As used herein, "complementary" in reference to an oligonucleotide means that at least 70% of the nucleobases of such oligonucleotide or one or more regions thereof and the nucleobases of another nucleic acid or one or more regions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions. Complementary nucleobases means nucleobases that are capable of forming hydrogen bonds with one another. Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5-methyl cytosine (.sup.mC) and guanine (G). Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. As used herein, "fully complementary" or "100% complementary" in reference to oligonucleotides means that such oligonucleotides are complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide.

[0019] As used herein, "conjugate group" means a group of atoms that is directly or indirectly attached to an oligonucleotide. Conjugate groups include a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.

[0020] As used herein, "conjugate linker" means a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide.

[0021] As used herein, "conjugate moiety" means a group of atoms that is attached to an oligonucleotide via a conjugate linker.

[0022] As used herein, "contiguous" in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other. For example, "contiguous nucleobases" means nucleobases that are immediately adjacent to each other in a sequence.

[0023] As used herein, "double-stranded antisense compound" means an antisense compound comprising two oligomeric compounds that are complementary to each other and form a duplex, and wherein one of the two said oligomeric compounds comprises an antisense oligonucleotide.

[0024] As used herein, "expanded repeat" in reference to a transcript or protein means a portion of a transcript or protein having a repeat region that has more repeats or repetitive elements than the corresponding repeat region of the corresponding wild type transcript or protein such that the number of repeats or repetitive elements in an "expanded repeat" transcript or protein is associated with a disease.

[0025] As used herein, "fully modified" in reference to a modified oligonucleotide means a modified oligonucleotide in which each sugar moiety is modified. "Uniformly modified" in reference to a modified oligonucleotide means a fully modified oligonucleotide in which each sugar moiety is the same. For example, the nucleosides of a uniformly modified oligonucleotide can each have a 2'-MOE modification but different nucleobase modifications, and the internucleoside linkages may be different.

[0026] As used herein, "FUS" means a FUS or TLS gene or a transcript or protein encoded by a FUS gene.

[0027] As used herein, "G3BP" means a G3BP stress granule assembly factor 1 gene or a transcript or protein encoded by a G3BP stress granule assembly factor 1 gene.

[0028] As used herein, "gapmer" means an antisense oligonucleotide comprising an internal "gap" region having a plurality of nucleosides that support RNase H cleavage positioned between external "wing" regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions.

[0029] As used herein, "hybridization" means the pairing or annealing of complementary oligonucleotides and/or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.

[0030] As used herein, "inhibiting formation" in reference to protein aggregates refers to a blockade or partial blockade of new protein aggregate formation and does not necessarily indicate a total elimination of new protein aggregate formation.

[0031] As used herein, the terms "internucleoside linkage" means a group or bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein "modified internucleoside linkage" means any internucleoside linkage other than a naturally occurring, phosphate internucleoside linkage. Non-phosphate linkages are referred to herein as modified internucleoside linkages. "Phosphorothioate linkage" means a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. A phosphorothioate internucleoside linkage is a modified internucleoside linkage. Modified internucleoside linkages include linkages that comprise abasic nucleosides. As used herein, "abasic nucleoside" means a sugar moiety in an oligonucleotide or oligomeric compound that is not directly connected to a nucleobase. In certain embodiments, an abasic nucleoside is adjacent to one or two nucleosides in an oligonucleotide.

[0032] As used herein, "linker-nucleoside" means a nucleoside that links, either directly or indirectly, an oligonucleotide to a conjugate moiety. Linker-nucleosides are located within the conjugate linker of an oligomeric compound. Linker-nucleosides are not considered part of the oligonucleotide portion of an oligomeric compound even if they are contiguous with the oligonucleotide.

[0033] As used herein, "low complexity domain" means a domain of a protein that is intrinsically disordered or lacking tertiary structure and comprises a low complexity sequence containing repeats of single amino acids or short amino acid motifs. In certain embodiments, low complexity domains are prio-like domains.

[0034] As used herein, "non-bicyclic modified sugar" or "non-bicyclic modified sugar moiety" means a modified sugar moiety that comprises a modification, such as a substitutent, that does not form a bridge between two atoms of the sugar to form a second ring.

[0035] As used herein, "linked nucleosides" are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked).

[0036] As used herein, "liquid miscibility" in reference to a protein or protein aggregate means the extent to which the protein or protein aggregate can mix with a liquid, as opposed to phase separate from said liquid. In certain embodiments, an increase in liquid miscibility of a protein or protein aggregate means that the protein or protein aggregate forms a more homogeneous mixture in the cytoplasm and decreases the extent to which it phase separates from the cytoplasm. In certain embodiments, an increase in liquid miscibility comprises an increase in water solubility.

[0037] As used herein, "messenger ribonucleoprotein complex" or "mRNP complex" means mRNA bound with proteins. As used herein, "messenger ribonucleoprotein granule" or "mRNP granule" means a protein aggregate comprising multiple mRNP complexes.

[0038] As used herein, "mismatch" or "non-complementary" means a nucleobase of a first oligonucleotide that is not complementary with the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligomeric compound are aligned.

[0039] As used herein, "modulation" means a perturbation of function, formation, activity, size, amount, or localization. Modulation of sub-cellular localization or distribution of a molecule means a change in a ratio of the amount of the molecule in two sub-cellular locations. Modulation of protein aggregation means a change in the function, formation, activity, size, amount, or localization of a protein aggregate or protein aggregates.

[0040] As used herein, "MOE" means methoxyethyl. "2'-MOE" means a 2'-OCH.sub.2CH.sub.2OCH.sub.3 group in place of the 2'-OH group of a ribosyl ring of a sugar moiety.

[0041] As used herein, "motif" means the pattern of unmodified and/or modified sugar moieties, nucleobases, and/or internucleoside linkages, in an oligonucleotide.

[0042] As used herein, "naturally occurring" means found in nature.

[0043] As used herein, "nucleobase" means a naturally occurring nucleobase or a modified nucleobase. As used herein a "naturally occurring nucleobase" is adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). As used herein, a modified nucleobase is a group of atoms capable of pairing with at least one naturally occurring nucleobase. A universal base is a nucleobase that can pair with any one of the five unmodified nucleobases. As used herein, "nucleobase sequence" means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar or internucleoside linkage modification.

[0044] As used herein, "nucleoside" means a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified. As used herein, "modified nucleoside" means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety.

[0045] As used herein, "oligomeric compound" means a compound consisting of an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group.

[0046] As used herein, "oligonucleotide" means a strand of linked nucleosides connected via internucleoside linkages, wherein each nucleoside and internucleoside linkage may be modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 8-50 linked nucleosides. As used herein, "modified oligonucleotide" means an oligonucleotide, wherein at least one nucleoside or internucleoside linkage is modified. As used herein, "unmodified oligonucleotide" means an oligonucleotide that does not comprise any nucleoside modifications or internucleoside modifications.

[0047] As used herein, "pharmaceutically acceptable carrier or diluent" means any substance suitable for use in administering to an animal. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile water; sterile saline; or sterile buffer solution.

[0048] As used herein "pharmaceutically acceptable salts" means physiologically and pharmaceutically acceptable salts of compounds, such as oligomeric compounds, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. As used herein "pharmaceutical composition" means a mixture of substances suitable for administering to a subject. For example, a pharmaceutical composition may comprise an antisense compound and a sterile aqueous solution. In certain embodiments, a pharmaceutical composition shows activity in free uptake assay in certain cell lines.

[0049] As used herein, "phosphorus moiety" means a group of atoms comprising a phosphorus atom. In certain embodiments, a phosphorus moiety comprises a mono-, di-, or tri-phosphate, or phosphorothioate.

[0050] As used herein, "processing body" means an mRNP granule that comprises RNA and at least one decapping factor or at least one protein that represses translation.

[0051] As used herein "prodrug" means a therapeutic agent in a form outside the body that is converted to a different form within the body or cells thereof. Typically conversion of a prodrug within the body is facilitated by the action of an enzymes (e.g., endogenous or viral enzyme) or chemicals present in cells or tissues and/or by physiologic conditions.

[0052] As used herein, "protein aggregate" means a complex comprising multiple protein molecules non-covalently bound together. In certain embodiments, protein aggregates comprise oligonucleotides and/or nucleic acids. As used herein, "reducing the size or amount of protein aggregates" means dissociating at least one component of a protein aggregate from the complex and/or reducing the number of protein aggregates present.

[0053] As used herein, "PSF" means a SFPQ or PSF gene, or a transcript or protein encoded by a SFPQ or PSF gene.

[0054] As used herein, "RAN translation product" or "repeat-associated non-ATG translation product" means a peptide or protein encoded by a portion of an RNA that contains a repeat region and lacks an AUG start codon. In certain embodiments, the repeat region is an expanded repeat.

[0055] As used herein, "RNA recognition motif" or "RRM" means a sequence or protein domain comprising at least one of the consensus sequences RNP1 and RNP2.

[0056] As used herein, "RNAi compound" means an antisense compound that acts, at least in part, through RISC or Ago2 to modulate a target nucleic acid and/or protein encoded by a target nucleic acid. RNAi compounds include, but are not limited to double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA, including microRNA mimics. In certain embodiments, an RNAi compound modulates the amount, activity, and/or splicing of a target nucleic acid. The term RNAi compound excludes antisense oligonucleotides that act through RNase H.

[0057] As used herein, the term "single-stranded" in reference to a compound means such a compound consisting of one oligomeric compound that is not paired with a second oligomeric compound to form a duplex. "Self-complementary" in reference to an oligonucleotide means an oligonucleotide that at least partially hybridizes to itself. A compound consisting of one oligomeric compound, wherein the oligonucleotide of the oligomeric compound is self-complementary, is a single-stranded compound. A single-stranded antisense or oligomeric compound may be capable of binding to a complementary oligomeric compound to form a duplex.

[0058] As used herein, "standard cell assay" means the assay described in Example X and reasonable variations thereof.

[0059] As used herein, "standard in vivo experiment" means the procedure described in Example X and reasonable variations thereof.

[0060] As used herein, "stress granule" means an mRNP granule that comprises components of the small ribosomal subunit, translation initiation factors, and/or poly(a)-binding protein. In certain embodiments, stress granules also contain G3BP.

[0061] As used herein, "sugar moiety" means an unmodified sugar moiety or a modified sugar moiety. As used herein, "unmodified sugar moiety" means a 2'-OH(H) ribosyl moiety, as found in RNA (an "unmodified RNA sugar moiety"), or a 2'-H(H) moiety, as found in DNA (an "unmodified DNA sugar moiety"). As used herein, "modified sugar moiety" or "modified sugar" means a modified furanosyl sugar moiety or a sugar surrogate. As used herein, modified furanosyl sugar moiety means a furanosyl sugar comprising a non-hydrogen substituent in place of at least one hydrogen of an unmodified sugar moiety. In certain embodiments, a modified furanosyl sugar moiety is a 2'-substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic sugars and non-bicyclic sugars. As used herein, "sugar surrogate" means a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group, such as an internucleoside linkage, conjugate group, or terminal group in an oligonucleotide. Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to complementary oligomeric compounds or nucleic acids.

[0062] As used herein, "target nucleic acid," "target RNA," "target RNA transcript" and "nucleic acid target" mean a nucleic acid that an antisense compound is designed to affect.

[0063] As used herein, "target region" means a portion of a target nucleic acid to which an antisense compound is designed to hybridize.

[0064] As used herein, "terminal group" means a chemical group or group of atoms that is covalently linked to a terminus of an oligonucleotide.

[0065] As used herein, "TDP-43" means a TAR DNA binding protein gene, or a transcript or protein encoded by a TAR DNA binding protein gene.

Certain Embodiments

[0066] The present disclosure includes but is not limited to the following embodiments.

[0067] Embodiment 1. A method of reducing the size or amount of protein aggregates in a cell comprising: contacting a cell with a compound comprising a modified oligonucleotide, thereby reducing the size or amount of protein aggregates in the cell.

[0068] Embodiment 2. A method of inhibiting the formation of protein aggregates in a cell comprising: contacting a cell with a compound comprising a modified oligonucleotide, thereby inhibiting the formation of protein aggregates in the cell.

[0069] Embodiment 3. A method of increasing liquid miscibility of a protein in a cell comprising: contacting a cell with a compound comprising a modified oligonucleotide, thereby increasing the liquid miscibility of a protein in the cell.

[0070] Embodiment 4. The method of embodiment 3, wherein the protein is in a protein aggregate.

[0071] Embodiment 5. The method of embodiment 3, wherein the liquid miscibility of the protein aggregate in the cell is increased.

[0072] Embodiment 6. The method of embodiment 4 or 5, wherein the size or amount of protein aggregates in the cell is reduced.

[0073] Embodiment 7. A method of modulating the sub-cellular distribution of at least one protein in a cell comprising: contacting a cell with a compound comprising a modified oligonucleotide, thereby modulating the sub-cellular distribution of at least one protein in the cell.

[0074] Embodiment 8. The method of embodiment 7, wherein the modulation of sub-cellular distribution of the at least one protein is an increase in the ratio of nuclear to cytoplasmic distribution of the at least one protein.

[0075] Embodiment 9. The method of embodiment 7 or 8, wherein the at least one protein is in a protein aggregate.

[0076] Embodiment 10. The method of embodiment 9, wherein the size or amount of protein aggregates in the cell is reduced.

[0077] Embodiment 11. The method of any of embodiments 1, 2, 4-6, 9, or 10, wherein the protein aggregates are present in the cytoplasm of the cell.

[0078] Embodiment 12. The method of embodiment 11, wherein the protein aggregates comprise an RNA-binding protein.

[0079] Embodiment 13. The method of embodiment 12, wherein the RNA-binding protein is FUS, TDP-43, or PSF.

[0080] Embodiment 14. The method of embodiment 12 or 13, wherein the RNA-binding protein comprises a mutation.

[0081] Embodiment 15. The method of embodiment 14, wherein the mutation is a point mutation.

[0082] Embodiment 16. The method of embodiment 14, wherein the mutation is an expanded repeat.

[0083] Embodiment 17. The method of embodiment 14, wherein the mutation is a deletion.

[0084] Embodiment 18. The method of any of embodiments 14-17, wherein the mutation causes protein aggregation, liquid immiscibility, and/or mislocalization of the protein in a cell.

[0085] Embodiment 19. The method of any of embodiments 12-18, wherein the RNA-binding protein comprises a low complexity domain.

[0086] Embodiment 20. The method of embodiment 19, wherein the modified oligonucleotide binds to the low complexity domain.

[0087] Embodiment 21. The method of any of embodiments 12-20, wherein the RNA-binding protein comprises an RNA recognition motif.

[0088] Embodiment 22. The method of embodiment 21, wherein the modified oligonucleotide does not bind to the RNA recognition motif.

[0089] Embodiment 23. The method of embodiment 21, wherein the modified oligonucleotide binds to the low complexity domain with higher affinity than it binds to the RNA recognition motif.

[0090] Embodiment 24. The method of any of embodiments 1-23, wherein the cell comprises a protein comprising an expanded repeat.

[0091] Embodiment 25. The method of any of embodiments 1-24, wherein the cell comprises Ran translation products.

[0092] Embodiment 26. The method of any of embodiments 1, 2, 4-6, or 9-25, wherein the protein aggregate is a messenger ribonucleoprotein granule.

[0093] Embodiment 27. The method of embodiment 26, wherein the protein aggregate is a stress granule

[0094] Embodiment 28. The method of embodiment 26, wherein the protein aggregate is processing body.

[0095] Embodiment 29. The method of any of embodiments 1, 2, 4-6, or 9-28, wherein the protein aggregate comprises G3BP protein.

[0096] Embodiment 30. The method of any of embodiments 1-29, wherein the modified oligonucleotide is a gapmer, wherein the gap consists of linked 2'-deoxynucleosides and the wings consist of linked nucleosides comprising modified sugar moieties.

[0097] Embodiment 31. The method of any of embodiments 1-30, wherein the modified oligonucleotide comprises at least one modified sugar moiety.

[0098] Embodiment 32. The method of embodiment 31, wherein the at least one modified sugar moiety is a cEt modified sugar moiety, a 2'-MOE modified sugar moiety, or a 2'-fluoro modified sugar moiety.

[0099] Embodiment 33. The method of embodiment 31, wherein the at least one modified sugar moiety is a 2'-fluoro modified sugar moiety.

[0100] Embodiment 34. The method of any of embodiments 1-33, wherein the modified oligonucleotide comprises at least one phosophorothioate internucleoside linkage.

[0101] Embodiment 35. The method of embodiment 34, wherein each internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage.

[0102] Embodiment 36. The method of any of embodiments 1-35, wherein the modified oligonucleotide comprises at least one modified nucleobase.

[0103] Embodiment 37. The method of embodiment 36, wherein the at least one modified nucleobase is a 5'-methyl cytosine.

[0104] Embodiment 38. The method of any of embodiments 1-37, wherein the nucleobase sequence of the modified oligonucleotide is not 100% complementary to a pre-mRNA or a mRNA in the cell.

[0105] Embodiment 39. The method of any of embodiments 1-38, wherein the compound comprises a conjugate group.

[0106] Embodiment 40. The method of any of embodiments 1-39, wherein the protein or protein aggregate is not a prion protein or prion protein aggregate.

[0107] Embodiment 41. The method of any of embodiments 1-40, wherein the cell is in an animal.

[0108] Embodiment 42. The method of any of embodiments 1-40, wherein the cell is in a human patient.

[0109] Embodiment 43. The method of embodiment 42, wherein the patient has a neurodegenerative disease.

[0110] Embodiment 44. The method of embodiment 42, wherein the patient has ALS.

[0111] Embodiment 45. The method of embodiment 42, wherein the patient has Alzheimer's Disease.

[0112] Embodiment 46. The method of embodiment 42, wherein the patient has juvenile onset ALS.

[0113] Embodiment 47. The method of embodiment 42, wherein the patient has Parkinson's Disease.

[0114] Embodiment 48. The method of embodiment 42, wherein the patient has frontotemporal dementia.

[0115] Embodiment 49. The method of embodiment 42, wherein the patient has Pick's Disease.

[0116] Embodiment 50. The method of any of embodiments 42-49, wherein at least one symptom in the patient is ameliorated.

[0117] Embodiment 51. The method of any of embodiments 42-50, wherein the patient's disease is treated or ameliorated.

[0118] Embodiment 52. The method of any of embodiments 1-51, comprising contacting a cell with a second compound comprising a modified oligonucleotide, wherein the second modified oligonucleotide is 100% complementary to a target nucleic acid in the cell.

[0119] Embodiment 53. The method of embodiment 52, wherein the target nucleic acid is a pre-mRNA or a mRNA.

[0120] Embodiment 54. A modified oligonucleotide for use in treating or ameliorating a neurodegenerative disease in a human in need thereof, wherein the modified oligonucleotide causes a reduction in the size or amount of cytoplasmic protein aggregates in the human.

[0121] Embodiment 55. Use of a modified oligonucleotide capable of causing a reduction in the size or amount of cytoplasmic protein aggregates in a cell for treatment of a neurodegenerative disease.

[0122] Embodiment 56. The method of any of embodiments 1-37 or 39-51, wherein the nucleobase sequence of the modified oligonucleotide is less than 70% complementary to a pre-mRNA or a mRNA in the cell.

[0123] Embodiment 57. A method of screening the sub-cellular distribution of at least one protein in a cell comprising: contacting a cell with a compound comprising a modified oligonucleotide and subsequently detecting the sub-cellular distribution of the at least one protein in the cell.

[0124] Embodiment 58. The method of embodiment 57, wherein the detection of the sub-cellular distribution of the at least one protein in the cell comprises contacting the cell with an antibody that binds to the at least one protein.

[0125] Embodiment 59. The method of embodiment 57 or 58, comprising contacting the cell with a vector that expresses a fusion protein prior to contacting the cell with the compound, wherein the fusion protein comprises a detectable tag.

[0126] Embodiment 60. The method of embodiment 59, wherein the detectable tag is a fluorescent protein.

[0127] Embodiment 61. The method of embodiment 59, wherein the detectable tag is an epitope tag.

[0128] Embodiment 62. The method of embodiment 60, wherein the fluorescent protein is a green fluorescent protein.

[0129] Embodiment 63. The method of any of embodiments 57-62, wherein the at least one protein is an RNA-binding protein.

[0130] Embodiment 64. The method of embodiment 63, wherein the RNA-binding protein is FUS, TDP-43, or PSF.

[0131] Embodiment 65. The method of embodiment 63 or 64, wherein the RNA-binding protein comprises a mutation.

[0132] Embodiment 66. The method of any of embodiments 59-65, wherein the fusion protein comprises FUS, TDP-43, or PSF.

I. Certain Oligonucleotides

[0133] In certain embodiments, the invention provides compounds that comprise or consist of oligonucleotides that consist of linked nucleosides. Oligonucleotides may be unmodified oligonucleotides (RNA or DNA) or may be modified oligonucleotides. Modified oligonucleotides comprise at least one modification relative to unmodified RNA or DNA (i.e., comprise at least one modified nucleoside (comprising a modified sugar moiety and/or a modified nucleobase) and/or at least one modified internucleoside linkage).

A. Certain Modified Nucleosides

[0134] Modified nucleosides comprise a modified sugar moiety or a modified nucleobase or both a modified sugar moiety and a modified nucleobase.

1. Certain Sugar Moieties

[0135] In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties.

[0136] In certain embodiments, modified sugar moieties are non-bicyclic modified furanosyl sugar moieties comprising one or more acyclic substituent, including but not limited to substituents at the 2', 4', and/or 5' positions. In certain embodiments, the furanosyl sugar moiety is a ribosyl sugar moiety. In certain embodiments one or more acyclic substituent of non-bicyclic modified sugar moieties is branched. Examples of 2'-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2'-F, 2'-OCH.sub.3 ("OMe" or "O-methyl"), and 2'-O(CH.sub.2).sub.2OCH.sub.3 ("MOE"). In certain embodiments, 2'-substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF.sub.3, OCF.sub.3, O--C.sub.1-C.sub.10 alkoxy, O--C.sub.1-C.sub.10 substituted alkoxy, O--C.sub.1-C.sub.10 alkyl, O--C.sub.1-C.sub.10 substituted alkyl, S-alkyl, N(R.sub.m)-alkyl, O-alkenyl, S-alkenyl, N(R.sub.m)-alkenyl, O-alkynyl, S-alkynyl, N(R.sub.m)-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ON(R.sub.m)(R.sub.n) or OCH.sub.2C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and R.sub.n is, independently, H, an amino protecting group, or substituted or unsubstituted C.sub.1-C.sub.10 alkyl, and the 2'-substituent groups described in Cook et al., U.S. Pat. No. 6,531,584; Cook et al., U.S. Pat. No. 5,859,221; and Cook et al., U.S. Pat. No. 6,005,087. Certain embodiments of these 2'-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO.sub.2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of 4'-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO 2015/106128. Examples of 5'-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In certain embodiments, non-bicyclic modified sugars comprise more than one non-bridging sugar substituent, for example, 2'-F-5'-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008/101157 and Rajeev et al., US2013/0203836.).

[0137] In certain embodiments, a 2'-substituted nucleoside or 2'-non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent group selected from: F, NH.sub.2, N.sub.3, OCF.sub.3, OCH.sub.3, O(CH.sub.2).sub.3NH.sub.2, CH.sub.2CH.dbd.CH.sub.2, OCH.sub.2CH.dbd.CH.sub.2, OCH.sub.2CH.sub.2OCH.sub.3, O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ON(R.sub.m)(R.sub.n), O(CH.sub.2).sub.2O(CH.sub.2).sub.2N(CH.sub.3).sub.2, and N-substituted acetamide (OCH.sub.2C(.dbd.O)--N(R.sub.m)(R.sub.n)), where each R.sub.m and R.sub.n is, independently, H, an amino protecting group, or substituted or unsubstituted C.sub.1-C.sub.10 alkyl.

[0138] In certain embodiments, a 2'-substituted nucleoside or 2'-non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent group selected from: F, OCF.sub.3, OCH.sub.3, OCH.sub.2CH.sub.2OCH.sub.3, O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ON(CH.sub.3).sub.2, O(CH.sub.2).sub.2O(CH.sub.2).sub.2N(CH.sub.3).sub.2, and OCH.sub.2C(.dbd.O)--N(H)CH.sub.3 ("NMA").

[0139] In certain embodiments, a 2'-substituted nucleoside or 2'-non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent group selected from: F, OCH.sub.3, and OCH.sub.2CH.sub.2OCH.sub.3.

[0140] Nucleosides comprising modified sugar moieties, such as non-bicyclic modified sugar moieties, may be referred to by the position(s) of the substitution(s) on the sugar moiety of the nucleoside. For example, nucleosides comprising 2'-substituted or 2-modified sugar moieties are referred to as 2'-substituted nucleosides or 2-modified nucleosides.

[0141] Certain modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms. In certain such embodiments, the furanose ring is a ribose ring. Examples of such 4' to 2' bridging sugar substituents include but are not limited to: 4'-CH.sub.2-2',4'-(CH.sub.2).sub.2-2', 4'-(CH.sub.2).sub.3-2', 4'-CH.sub.2--O-2' ("LNA"), 4'-CH.sub.2--S-2', 4'-(CH.sub.2).sub.2--O-2' ("ENA"), 4'-CH(CH.sub.3)--O-2' (referred to as "constrained ethyl" or "cEt" when in the S configuration), 4'-CH.sub.2--O--CH.sub.2-2', 4'-CH.sub.2--N(R)-2', 4'-CH(CH.sub.2OCH.sub.3)--O-2' ("constrained MOE" or "cMOE") and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 7,399,845, Bhat et al., U.S. Pat. No. 7,569,686, Swayze et al., U.S. Pat. No. 7,741,457, and Swayze et al., U.S. Pat. No. 8,022,193), 4'-C(CH.sub.3)(CH.sub.3)--O-2' and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 8,278,283), 4'-CH.sub.2--N(OCH.sub.3)-2' and analogs thereof (see, e.g., Prakash et al., U.S. Pat. No. 8,278,425), 4'-CH.sub.2--O--N(CH.sub.3)-2' (see, e.g., Allerson et al., U.S. Pat. No. 7,696,345 and Allerson et al., U.S. Pat. No. 8,124,745), 4'-CH.sub.2--C(H)(CH.sub.3)-2' (see, e.g., Zhou, et al., J. Org. Chem., 2009, 74, 118-134), 4'-CH.sub.2--C(.dbd.CH.sub.2)-2' and analogs thereof (see e.g., Seth et al., U.S. Pat. No. 8,278,426), 4'-C(R.sub.aR.sub.b)--N(R)--O-2', 4'-C(R.sub.aR.sub.b)--O--N(R)-2', 4'-CH.sub.2--O--N(R)-2', and 4'-CH.sub.2--N(R)--O-2', wherein each R, R.sub.a, and R.sub.b is, independently, H, a protecting group, or C.sub.1-C.sub.12 alkyl (see, e.g. Imanishi et al., U.S. Pat. No. 7,427,672).

[0142] In certain embodiments, such 4' to 2' bridges independently comprise from 1 to 4 linked groups independently selected from: --[C(R.sub.a)(R.sub.b)].sub.n--, --[C(R.sub.a)(R.sub.b)].sub.n--O--, --C(R.sub.a).dbd.C(R.sub.b)--, --C(R.sub.a).dbd.N--, --C(.dbd.NR.sub.a)--, --C(.dbd.O)--, --C(.dbd.S)--, --O--, --Si(R.sub.a).sub.2--, --S(.dbd.O).sub.x--, and --N(R.sub.a)--;

[0143] wherein:

[0144] x is 0, 1, or 2;

[0145] n is 1, 2, 3, or 4;

[0146] each R.sub.a and R.sub.b is, independently, H, a protecting group, hydroxyl, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C.sub.5-C.sub.7 alicyclic radical, substituted C.sub.5-C.sub.7 alicyclic radical, halogen, OJ.sub.1, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1, acyl (C(.dbd.O)--H), substituted acyl, CN, sulfonyl (S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and

[0147] each J.sub.1 and J.sub.2 is, independently, H, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, acyl (C(.dbd.O)--H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C.sub.1-C.sub.12 aminoalkyl, substituted C.sub.1-C.sub.12 aminoalkyl, or a protecting group.

[0148] Additional bicyclic sugar moieties are known in the art, see, for example: Freier et al., Nucleic Acids Research, 1997, 25 (22), 4429-4443, Albaek et al., J. Org. Chem., 2006, 71, 7731-7740, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 20017, 129, 8362-8379; Elayadi et al., Wengel et al., U.S. Pat. No. 7,053,207; Imanishi et al., U.S. Pat. No. 6,268,490; Imanishi et al. U.S. Pat. No. 6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al., U.S. Pat. No. 6,794,499; Wengel et al., U.S. Pat. No. 6,670,461; Wengel et al., U.S. Pat. No. 7,034,133; Wengel et al., U.S. Pat. No. 8,080,644; Wengel et al., U.S. Pat. No. 8,034,909; Wengel et al., U.S. Pat. No. 8,153,365; Wengel et al., U.S. Pat. No. 7,572,582; and Ramasamy et al., U.S. Pat. No. 6,525,191;; Torsten et al., WO 2004/106356;Wengel et al., WO 1999/014226; Seth et al., WO 2007/134181; Seth et al., U.S. Pat. No. 7,547,684; Seth et al., U.S. Pat. No. 7,666,854; Seth et al., U.S. Pat. No. 8,088,746; Seth et al., U.S. Pat. No. 7,750,131; Seth et al., U.S. Pat. No. 8,030,467; Seth et al., U.S. Pat. No. 8,268,980; Seth et al., U.S. Pat. No. 8,546,556; Seth et al., U.S. Pat. No. 8,530,640; Migawa et al., U.S. Pat. No. 9,012,421; Seth et al., U.S. Pat. No. 8,501,805; and U.S. Patent Publication Nos. Allerson et al., US2008/0039618 and Migawa et al., US2015/0191727.

[0149] In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, an LNA nucleoside (described herein) may be in the .alpha.-L configuration or in the .beta.-D configuration.

##STR00001##

.alpha.-L-methyleneoxy (4'-CH.sub.2--O-2') or .alpha.-L-LNA bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). Herein, general descriptions of bicyclic nucleosides include both isomeric configurations. When the positions of specific bicyclic nucleosides (e.g., LNA or cEt) are identified in exemplified embodiments herein, they are in the .beta.-D configuration, unless otherwise specified.

[0150] In certain embodiments, modified sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5'-substituted and 4'-2' bridged sugars).

[0151] In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moieties also comprise bridging and/or non-bridging substituents as described herein. For example, certain sugar surrogates comprise a 4'-sulfur atom and a substitution at the 2'-position (see, e.g., Bhat et al., U.S. Pat. No. 7,875,733 and Bhat et al., U.S. Pat. No. 7,939,677) and/or the 5' position.

[0152] In certain embodiments, sugar surrogates comprise rings having other than 5 atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran ("THP"). Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include but are not limited to hexitol nucleic acid ("HNA"), anitol nucleic acid ("ANA"), manitol nucleic acid ("MNA") (see, e.g., Leumann, C J. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoro HNA:

##STR00002##

("F-HNA", see e.g. Swayze et al., U.S. Pat. No. 8,088,904; Swayze et al., U.S. Pat. No. 8,440,803; Swayze et al., U.S. Pat. No. 8,796,437; and Swayze et al., U.S. Pat. No. 9,005,906; F-HNA can also be referred to as a F-THP or 3'-fluoro tetrahydropyran), and nucleosides comprising additional modified THP compounds having the formula:

##STR00003##

wherein, independently, for each of said modified THP nucleoside:

[0153] Bx is a nucleobase moiety;

[0154] T.sub.3 and T.sub.4 are each, independently, an internucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide or one of T.sub.3 and T.sub.4 is an internucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide and the other of T.sub.3 and T.sub.4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3'-terminal group; q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each, independently, H, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, or substituted C.sub.2-C.sub.6 alkynyl; and

[0155] each of R.sub.1 and R.sub.2 is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, OC(.dbd.X)J.sub.1, OC(.dbd.X)NJ.sub.1J.sub.2, NJ.sub.3C(.dbd.X)NJ.sub.1J.sub.2, and CN, wherein X is O, S or NJ.sub.1, and each J.sub.1, J.sub.2, and J.sub.3 is, independently, H or C.sub.1-C.sub.6 alkyl.

[0156] In certain embodiments, modified THP nucleosides are provided wherein q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each H. In certain embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 is other than H. In certain embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 is methyl. In certain embodiments, modified THP nucleosides are provided wherein one of R.sub.1 and R.sub.2 is F. In certain embodiments, R.sub.1 is F and R.sub.2 is H, in certain embodiments, R.sub.1 is methoxy and R.sub.2 is H, and in certain embodiments, R.sub.1 is methoxyethoxy and R.sub.2 is H.

[0157] In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. Pat. No. 5,698,685; Summerton et al., U.S. Pat. No. 5,166,315; Summerton et al., U.S. Pat. No. 5,185,444; and Summerton et al., U.S. Pat. No. 5,034,506). As used here, the term "morpholino" means a sugar surrogate having the following structure:

##STR00004##

In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as "modified morpholinos."

[0158] In certain embodiments, sugar surrogates comprise acyclic moieties. Examples of nucleosides and oligonucleotides comprising such acyclic sugar surrogates include but are not limited to: peptide nucleic acid ("PNA"), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and nucleosides and oligonucleotides described in Manoharan et al., WO2011/133876.

[0159] Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used in modified nucleosides).

2. Certain Modified Nucleobases

[0160] In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising an unmodified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase.

[0161] In certain embodiments, modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines. In certain embodiments, modified nucleobases are selected from: 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine , 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (--C.ident.C--CH.sub.3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in Merigan et al., U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442-443.

[0162] Publications that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, Manohara et al., US2003/0158403; Manoharan et al., US2003/0175906;; Dinh et al., U.S. Pat. No. 4,845,205; Spielvogel et al., U.S. Pat. No. 5,130,302; Rogers et al., U.S. Pat. No. 5,134,066; Bischofberger et al., U.S. Pat. No. 5,175,273; Urdea et al., U.S. Pat. No. 5,367,066; Benner et al., U.S. Pat. No. 5,432,272; Matteucci et al., U.S. Pat. No. 5,434,257; Gmeiner et al., U.S. Pat. No. 5,457,187; Cook et al., U.S. Pat. No. 5,459,255; Froehler et al., U.S. Pat. No. 5,484,908; Matteucci et al., U.S. Pat. No. 5,502,177; Hawkins et al., U.S. Pat. No. 5,525,711; Haralambidis et al., U.S. Pat. No. 5,552,540; Cook et al., U.S. Pat. No. 5,587,469; Froehler et al., U.S. Pat. No. 5,594,121; Switzer et al., U.S. Pat. No. 5,596,091; Cook et al., U.S. Pat. No. 5,614,617; Froehler et al., U.S. Pat. No. 5,645,985; Cook et al., U.S. Pat. No. 5,681,941; Cook et al., U.S. Pat. No. 5,811,534; Cook et al., U.S. Pat. No. 5,750,692; Cook et al., U.S. Pat. No. 5,948,903; Cook et al., U.S. Pat. No. 5,587,470; Cook et al., U.S. Pat. No. 5,457,191; Matteucci et al., U.S. Pat. No. 5,763,588; Froehler et al., U.S. Pat. No. 5,830,653; Cook et al., U.S. Pat. No. 5,808,027; Cook et al., U.S. Pat. No. 6,166,199; and Matteucci et al., U.S. Pat. No. 6,005,096.

B. Certain Modified Internucleoside Linkages

[0163] In certain embodiments, nucleosides of modified oligonucleotides may be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include but are not limited to phosphates, which contain a phosphodiester bond ("P.dbd.O") (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates ("P.dbd.S"), and phosphorodithioates ("HS--P.dbd.S"). Representative non-phosphorus containing internucleoside linking groups include but are not limited to methylenemethylimino (--CH.sub.2--N(CH.sub.3)--O--CH.sub.2--), thiodiester, thionocarbamate (--O--C(.dbd.O)(NH)--S--); siloxane (--O--SiH.sub.2--O--); and N,N'-dimethylhydrazine (--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--). Modified internucleoside linkages, compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral internucleoside linkages include but are not limited to alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.

[0164] Neutral internucleoside linkages include, without limitation, phosphotriesters, methylphosphonates, MMI (3'-CH.sub.2--N(CH.sub.3)--O-5'), amide-3 (3'-CH.sub.2--C(.dbd.O)--N(H)-5'), amide-4 (3'-CH.sub.2--N(H)--C(.dbd.O)-5'), formacetal (3'-O--CH.sub.2--O-5'), methoxypropyl, and thioformacetal (3'-S--CH.sub.2--O-5'). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH.sub.2 component parts.

C. Certain Motifs

[0165] In certain embodiments, modified oligonucleotides comprise one or more modified nucleoside comprising a modified sugar. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified internucleoside linkage. In such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and/or internucleoside linkages of a modified oligonucleotide define a pattern or motif. In certain embodiments, the patterns or motifs of sugar moieties, nucleobases, and internucleoside linkages are each independent of one another. Thus, a modified oligonucleotide may be described by its sugar motif, nucleobase motif and/or internucleoside linkage motif (as used herein, nucleobase motif describes the modifications to the nucleobases independent of the nucleobase sequence).

1. Certain Sugar Motifs

[0166] In certain embodiments, oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif. In certain instances, such sugar motifs include but are not limited to any of the sugar modifications discussed herein.

[0167] In certain embodiments, modified oligonucleotides comprise or consist of a region having a gapmer motif, which comprises two external regions or "wings" and a central or internal region or "gap." The three regions of a gapmer motif (the 5'-wing, the gap, and the 3'-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3'-most nucleoside of the 5'-wing and the 5'-most nucleoside of the 3'-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap (i.e., the wing/gap junction). In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar motif of the 5'-wing differs from the sugar motif of the 3'-wing (asymmetric gapmer).

[0168] In certain embodiments, the wings of a gapmer comprise 1-5 nucleosides. In certain embodiments, the wings of a gapmer comprise 2-5 nucleosides. In certain embodiments, the wings of a gapmer comprise 3-5 nucleosides. In certain embodiments, the nucleosides of a gapmer are all modified nucleosides.

[0169] In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides. In certain embodiments, the gap of a gapmer comprises 7-10 nucleosides. In certain embodiments, the gap of a gapmer comprises 8-10 nucleosides. In certain embodiments, the gap of a gapmer comprises 10 nucleosides. In certain embodiment, each nucleoside of the gap of a gapmer is an unmodified 2'-deoxy nucleoside.

[0170] In certain embodiments, the nucleosides on the gap side of each wing/gap junction are unmodified 2'-deoxyribosyl nucleosides and the nucleosides on the wing sides of each wing/gap junction are modified nucleosides. In certain such embodiments, each nucleoside of the gap is an unmodified 2'-deoxyribosyl nucleoside. In certain such embodiments, each nucleoside of each wing is a modified nucleoside.

[0171] In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif. In such embodiments, each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety. In certain such embodiments, each nucleoside to the entire modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, a fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of a uniformly modified comprises the same 2'-modification.

2. Certain Nucleobase Motifs

[0172] In certain embodiments, oligonucleotides comprise modified and/or unmodified nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases are modified. In certain embodiments, each purine or each pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all of the cytosine nucleobases in a modified oligonucleotide are 5-methylcytosines.

[0173] In certain embodiments, modified oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3'-end of the oligonucleotide. In certain embodiments the block is within 3 nucleosides of the 3'-end of the oligonucleotide. In certain embodiments, the block is at the 5'-end of the oligonucleotide. In certain embodiments the block is within 3 nucleosides of the 5'-end of the oligonucleotide.

[0174] In certain embodiments, oligonucleotides having a gapmer motif comprise a nucleoside comprising a modified nucleobase. In certain such embodiments, one nucleoside comprising a modified nucleobase is in the central gap of an oligonucleotide having a gapmer motif. In certain such embodiments, the sugar moiety of said nucleoside is a 2'-deoxyribosyl moiety. In certain embodiments, the modified nucleobase is selected from: a 2-thiopyrimidine and a 5-propynepyrimidine.

3. Certain Internucleoside Linkage Motifs

[0175] In certain embodiments, oligonucleotides comprise modified and/or unmodified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, essentially each internucleoside linking group is a phosphate internucleoside linkage (P.dbd.O). In certain embodiments, each internucleoside linking group of a modified oligonucleotide is a phosphorothioate (P.dbd.S). In certain embodiments, each internucleoside linking group of a modified oligonucleotide is independently selected from a phosphorothioate and phosphate internucleoside linkage. In certain embodiments, the sugar motif of a modified oligonucleotide is a gapmer and the internucleoside linkages within the gap are all modified. In certain such embodiments, some or all of the internucleoside linkages in the wings are unmodified phosphate linkages. In certain embodiments, the terminal internucleoside linkages are modified.

D. Certain Lengths

[0176] In certain embodiments, oligonucleotides (including modified oligonucleotides) can have any of a variety of ranges of lengths. In certain embodiments, oligonucleotides consist of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X.ltoreq.Y. For example, in certain embodiments, oligonucleotides consist of 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides

E. Certain Modified Oligonucleotides

[0177] In certain embodiments, the above modifications (sugar, nucleobase, internucleoside linkage) are incorporated into a modified oligonucleotide. In certain embodiments, modified oligonucleotides are characterized by their modification motifs and overall lengths. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. For example, the internucleoside linkages within the wing regions of a sugar gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region of the sugar motif. Likewise, such sugar gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. Furthermore, in certain instances, an oligonucleotide is described by an overall length or range and by lengths or length ranges of two or more regions (e.g., regions of nucleosides having specified sugar modifications), in such circumstances it may be possible to select numbers for each range that result in an oligonucleotide having an overall length falling outside the specified range. In such circumstances, both elements must be satisfied. For example, in certain embodiments, a modified oligonucleotide consists if of 15-20 linked nucleosides and has a sugar motif consisting of three regions, A, B, and C, wherein region A consists of 2-6 linked nucleosides having a specified sugar motif, region B consists of 6-10 linked nucleosides having a specified sugar motif, and region C consists of 2-6 linked nucleosides having a specified sugar motif. Such embodiments do not include modified oligonucleotides where A and C each consist of 6 linked nucleosides and B consists of 10 linked nucleosides (even though those numbers of nucleosides are permitted within the requirements for A, B, and C) because the overall length of such oligonucleotide is 22, which exceeds the upper limit of the overall length of the modified oligonucleotide (20). Herein, if a description of an oligonucleotide is silent with respect to one or more parameter, such parameter is not limited. Thus, a modified oligonucleotide described only as having a gapmer sugar motif without further description may have any length, internucleoside linkage motif, and nucleobase motif. Unless otherwise indicated, all modifications are independent of nucleobase sequence.

F. Nucleobase Sequence

[0178] In certain embodiments, oligonucleotides (unmodified or modified oligonucleotides) are further described by their nucleobase sequence. In certain embodiments oligonucleotides have a nucleobase sequence that is complementary to a second oligonucleotide or a target nucleic acid. In certain such embodiments, a region of an oligonucleotide has a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain embodiments, the nucleobase sequence of a region or entire length of an oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% complementary to the second oligonucleotide or nucleic acid, such as a target nucleic acid.

[0179] In certain embodiments, oligonucleotides have a nucleobase sequence that is not 100% complementary to a target nucleic acid or any nucleic acid in a cell. In certain such embodiments, oligonucleotides are less than 90% complementary to any nucleic acid in a cell. In certain such embodiments, oligonucleotides are less than 80% or less than 70% complementary to any nucleic acid in a cell. In certain embodiments, oligonucleotides have a nucleobase sequence that is less than 100%, less than 90%, less than 80%, or less than 70% complementary to any known nucleic acid sequence in the cell.

[0180] In certain embodiments, methods described herein comprise contacting a cell with a first compound comprising a first modified oligonucleotide and a second compound comprising a second modified oligonucleotide, wherein the nucleobase sequence of one of the first and second modified oligonucleotides is complementary to a target nucleic acid and the nucleobase sequence of the other of the first and second modified oligonucleotides is less than 100%, less than 90%, less than 80%, or less than 70% complementary to any target nucleic acid or any nucleic acid in the cell. In certain such embodiments, the modified oligonucleotide that is less than 100%, less than 90%, less than 80%, or less than 70% complementary to any target nucleic acid or any nucleic acid in the cell modulates protein aggregation and/or sub-cellular distribution of at least one protein. In certain such embodiments, the size or amount of protein aggregates in the cell is decreased and/or the nuclear to cytoplasmic ratio of the sub-cellular distribution of the at least one protein is increased.

II. Certain Oligomeric Compounds

[0181] In certain embodiments, the invention provides oligomeric compounds, which consist of an oligonucleotide (modified or unmodified) and optionally one or more conjugate groups and/or terminal groups. Conjugate groups consist of one or more conjugate moiety and a conjugate linker which links the conjugate moiety to the oligonucleotide. Conjugate groups may be attached to either or both ends of an oligonucleotide and/or at any internal position. In certain embodiments, conjugate groups are attached to the 2'-position of a nucleoside of a modified oligonucleotide. In certain embodiments, conjugate groups that are attached to either or both ends of an oligonucleotide are terminal groups. In certain such embodiments, conjugate groups or terminal groups are attached at the 3' and/or 5'-end of oligonucleotides. In certain such embodiments, conjugate groups (or terminal groups) are attached at the 3'-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 3'-end of oligonucleotides. In certain embodiments, conjugate groups (or terminal groups) are attached at the 5'-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 5'-end of oligonucleotides.

[0182] Examples of terminal groups include but are not limited to conjugate groups, capping groups, phosphate moieties, protecting groups, abasic nucleosides, modified or unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.

A. Certain Conjugate Groups

[0183] In certain embodiments, oligonucleotides are covalently attached to one or more conjugate groups. In certain embodiments, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance. In certain embodiments, conjugate groups impart a new property on the attached oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide. Certain conjugate groups and conjugate moieties have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), a tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or a GalNAc cluster (e.g., WO2014/179620).

1. Conjugate Moieties

[0184] Conjugate moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates (e.g., GalNAc), vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

[0185] In certain embodiments, a conjugate moiety comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

2. Conjugate Linkers

[0186] Conjugate moieties are attached to oligonucleotides through conjugate linkers. In certain compounds comprising oligonucleotides, such as oligomeric compounds, the conjugate linker is a single chemical bond (i.e., the conjugate moiety is attached directly to an oligonucleotide through a single bond). In certain oligomeric compounds, a conjugate moiety is attached to an oligonucleotide via a more complex conjugate linker comprising one or more conjugate linker moieties, which are sub-units making up a conjugate linker. In certain embodiments, the conjugate linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units such as ethylene glycol, nucleosides, or amino acid units.

[0187] In certain embodiments, a conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises groups selected from alkyl, amino, oxo, amide and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker includes at least one neutral linking group.

[0188] In certain embodiments, conjugate linkers, including the conjugate linkers described above, are bifunctional linking moieties, e.g., those known in the art to be useful for attaching conjugate groups to parent compounds, such as the oligonucleotides provided herein. In general, a bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected to bind to a particular site on a parent compound and the other is selected to bind to a conjugate group. Examples of functional groups used in a bifunctional linking moiety include but are not limited to electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.

[0189] Examples of conjugate linkers include but are not limited to pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include but are not limited to substituted or unsubstituted C.sub.1-C.sub.10 alkyl, substituted or unsubstituted C.sub.2-C.sub.10 alkenyl or substituted or unsubstituted C.sub.2-C.sub.10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

[0190] In certain embodiments, conjugate linkers comprise 1-10 linker-nucleosides In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments such linker-nucleosides comprise a modified sugar moiety. In certain embodiments, linker-nucleosides are unmodified. In certain embodiments, linker-nucleosides comprise an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, a cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typically desirable for linker-nucleosides to be cleaved from the oligomeric compound after it reaches a target tissue. Accordingly, linker-nucleosides are typically linked to one another and to the remainder of the oligomeric compound through cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.

[0191] Herein, linker-nucleosides are not considered to be part of the oligonucleotide. Accordingly, in embodiments in which an oligomeric compound comprises an oligonucleotide consisting of a specified number or range of linked nucleosides and/or a specified percent complementarity to a reference nucleic acid and the oligomeric compound also comprises a conjugate group comprising a conjugate linker comprising linker-nucleosides, those linker-nucleosides are not counted toward the length of the oligonucleotide and are not used in determining the percent complementarity of the oligonucleotide for the reference nucleic acid. For example, an oligomeric compound may comprise (1) a modified oligonucleotide consisting of 8-30 nucleosides and (2) a conjugate group comprising 1-10 linker-nucleosides that are contiguous with the nucleosides of the modified oligonucleotide. The total number of contiguous linked nucleosides in such an oligomeric compound is more than 30. Alternatively, an oligomeric compound may comprise a modified oligonucleotide consisting of 8-30 nucleosides and no conjugate group. The total number of contiguous linked nucleosides in such an oligomeric compound is no more than 30. Unless otherwise indicated conjugate linkers comprise no more than 10 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 5 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 2 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 1 linker-nucleoside.

[0192] In certain embodiments, it is desirable for a conjugate group to be cleaved from the oligonucleotide. For example, in certain circumstances oligomeric compounds comprising a particular conjugate moiety are better taken up by a particular cell type, but once the oligomeric compound has been taken up, it is desirable that the conjugate group be cleaved to release the unconjugated or parent oligonucleotide. Thus, certain conjugate linkers may comprise one or more cleavable moieties. In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety is a group of atoms comprising at least one cleavable bond. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, a cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome. In certain embodiments, a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.

[0193] In certain embodiments, a cleavable bond is selected from among: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, a cleavable bond is one or both of the esters of a phosphodiester. In certain embodiments, a cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a phosphate linkage between an oligonucleotide and a conjugate moiety or conjugate group.

[0194] In certain embodiments, a cleavable moiety comprises or consists of one or more linker-nucleosides. In certain such embodiments, the one or more linker-nucleosides are linked to one another and/or to the remainder of the oligomeric compound through cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, a cleavable moiety is 2'-deoxy nucleoside that is attached to either the 3' or 5'-terminal nucleoside of an oligonucleotide by a phosphate internucleoside linkage and covalently attached to the remainder of the conjugate linker or conjugate moiety by a phosphate or phosphorothioate linkage. In certain such embodiments, the cleavable moiety is 2'-deoxyadenosine.

[0195] In certain embodiments, compounds of the invention are single-stranded. In certain embodiments, oligomeric compounds are paired with a second oligonucleotide or oligomeric compound to form a duplex, which is double-stranded.

III. Certain Antisense Compounds

[0196] In certain embodiments, the present invention provides antisense compounds, which comprise or consist of an oligomeric compound comprising an antisense oligonucleotide, having a nucleobase sequences complementary to that of a target nucleic acid. In certain embodiments, antisense compounds are single-stranded. Such single-stranded antisense compounds typically comprise or consist of an oligomeric compound that comprises or consists of a modified oligonucleotide and optionally a conjugate group. In certain embodiments, antisense compounds are double-stranded. Such double-stranded antisense compounds comprise a first oligomeric compound having a region complementary to a target nucleic acid and a second oligomeric compound having a region complementary to the first oligomeric compound. The first oligomeric compound of such double stranded antisense compounds typically comprises or consists of a modified oligonucleotide and optionally a conjugate group. The oligonucleotide of the second oligomeric compound of such double-stranded antisense compound may be modified or unmodified. Either or both oligomeric compounds of a double-stranded antisense compound may comprise a conjugate group. The oligomeric compounds of double-stranded antisense compounds may include non-complementary overhanging nucleosides.

[0197] In certain embodiments, oligomeric compounds of antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, antisense compounds selectively affect one or more target nucleic acid. Such selective antisense compounds comprise a nucleobase sequence that hybridizes to one or more target nucleic acid, resulting in one or more desired antisense activity and does not hybridize to one or more non-target nucleic acid or does not hybridize to one or more non-target nucleic acid in such a way that results in significant undesired antisense activity.

[0198] In certain antisense activities, hybridization of an antisense compound to a target nucleic acid results in recruitment of a protein that cleaves the target nucleic acid. For example, certain antisense compounds result in RNase H mediated cleavage of the target nucleic acid. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. The DNA in such an RNA:DNA duplex need not be unmodified DNA. In certain embodiments, the invention provides antisense compounds that are sufficiently "DNA-like" to elicit RNase H activity. Further, in certain embodiments, one or more non-DNA-like nucleoside in the gap of a gapmer is tolerated.

[0199] In certain antisense activities, an antisense compound or a portion of an antisense compound is loaded into an RNA-induced silencing complex (RISC), ultimately resulting in cleavage of the target nucleic acid. For example, certain antisense compounds result in cleavage of the target nucleic acid by Argonaute. Antisense compounds that are loaded into RISC are RNAi compounds. RNAi compounds may be double-stranded (siRNA) or single-stranded (ssRNA).

[0200] In certain embodiments, hybridization of an antisense compound to a target nucleic acid does not result in recruitment of a protein that cleaves that target nucleic acid. In certain such embodiments, hybridization of the antisense compound to the target nucleic acid results in alteration of splicing of the target nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid. In certain such embodiments, hybridization of an antisense compound to a target nucleic acid results in alteration of translation of the target nucleic acid.

[0201] Antisense activities may be observed directly or indirectly. In certain embodiments, observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid, a change in the ratio of splice variants of a nucleic acid or protein, and/or a phenotypic change in a cell or animal.

IV. Certain Target Nucleic Acids

[0202] In certain embodiments, antisense compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from: an mRNA and a pre-mRNA, including intronic, exonic and untranslated regions. In certain embodiments, the target RNA is an mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain such embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron.

[0203] In certain embodiments, the target nucleic acid is a non-coding RNA. In certain such embodiments, the target non-coding RNA is selected from: a long-non-coding RNA, a short non-coding RNA, an intronic RNA molecule, a snoRNA, a scaRNA, a microRNA (including pre-microRNA and mature microRNA), a ribosomal RNA, and promoter directed RNA. In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA. In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA or a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA or an intronic region of a pre-mRNA. In certain embodiments, the target nucleic acid is a long non-coding RNA. In certain embodiments, the target nucleic acid is a non-coding RNA associated with splicing of other pre-mRNAs. In certain embodiments, the target nucleic acid is a nuclear-retained non-coding RNA.

[0204] In certain embodiments, antisense compounds described herein are complementary to a target nucleic acid comprising a single-nucleotide polymorphism (SNP). In certain such embodiments, the antisense compound is capable of modulating expression of one allele of the SNP-containing target nucleic acid to a greater or lesser extent than it modulates another allele. In certain embodiments, an antisense compound hybridizes to a (SNP)-containing target nucleic acid at the single-nucleotide polymorphism site.

[0205] In certain embodiments, antisense compounds are at least partially complementary to more than one target nucleic acid. For example, antisense compounds of the present invention may mimic microRNAs, which typically bind to multiple targets.

A. Complementarity/Mismatches to the Target Nucleic Acid

[0206] In certain embodiments, antisense compounds comprise antisense oligonucleotides that are complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, such oligonucleotides are 99% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 95% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 90% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, antisense oligonucleotides are at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide and comprise a region that is 100% or fully complementary to a target nucleic acid. In certain such embodiments, the region of full complementarity is from 6 to 20 nucleobases in length. In certain such embodiments, the region of full complementarity is from 10 to 18 nucleobases in length. In certain such embodiments, the region of full complementarity is from 18 to 20 nucleobases in length.

[0207] In certain embodiments, oligonucleotides comprise one or more mismatched nucleobases relative to the target nucleic acid. In certain such embodiments, antisense activity against the target is reduced by such mismatch, but activity against a non-target is reduced by a greater amount. Thus, in certain such embodiments selectivity of the antisense compound is improved. In certain embodiments, the mismatch is specifically positioned within an oligonucleotide having a gapmer motif. In certain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, or 8 from the 5'-end of the gap region. In certain such embodiments, the mismatch is at position 9, 8, 7, 6, 5, 4, 3, 2, 1 from the 3'-end of the gap region. In certain such embodiments, the mismatch is at position 1, 2, 3, or 4 from the 5'-end of the wing region. In certain such embodiments, the mismatch is at position 4, 3, 2, or 1 from the 3'-end of the wing region.

V. Certain Pharmaceutical Compositions

[0208] In certain embodiments, the present invention provides pharmaceutical compositions comprising one or more antisense compound or a salt thereof. In certain such embodiments, the pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more antisense compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more antisense compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and sterile water. In certain embodiments, a pharmaceutical composition consists of one antisense compound and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile PBS. In certain embodiments, the sterile PBS is pharmaceutical grade PBS.

[0209] In certain embodiments, pharmaceutical compositions comprise one or more or antisense compound and one or more excipients. In certain such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

[0210] In certain embodiments, antisense compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

[0211] In certain embodiments, pharmaceutical compositions comprising an antisense compound encompass any pharmaceutically acceptable salts of the antisense compound, esters of the antisense compound, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising antisense compounds comprising one or more antisense oligonucleotide, upon administration to an animal, including a human, are capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In certain embodiments, prodrugs comprise one or more conjugate group attached to an oligonucleotide, wherein the conjugate group is cleaved by endogenous nucleases within the body.

[0212] Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid, such as an antisense compound, is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.

[0213] In certain embodiments, pharmaceutical compositions comprise a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

[0214] In certain embodiments, pharmaceutical compositions comprise one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

[0215] In certain embodiments, pharmaceutical compositions comprise a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80.TM. and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80.TM.; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

[0216] In certain embodiments, pharmaceutical compositions are prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration. In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain.

Nonlimiting Disclosure and Incorporation by Reference

[0217] While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, and other publications recited in the present application is incorporated herein by reference in its entirety.

[0218] Although the sequence listing accompanying this filing identifies each sequence as either "RNA" or "DNA" as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as "RNA" or "DNA" to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2'-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2'-OH in place of one 2'-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) in place of a uracil of RNA). Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligomeric compound having the nucleobase sequence "ATCGATCG" encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence "AUCGAUCG" and those having some DNA bases and some RNA bases such as "AUCGATCG" and oligomeric compounds having other modified nucleobases, such as "AT.sup.mCGAUCG," wherein .sup.mC indicates a cytosine base comprising a methyl group at the 5-position.

[0219] Certain compounds described herein (e.g., modified oligonucleotides) have one or more asymmetric center and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), as .alpha. or .beta. such as for sugar anomers, or as (D) or (L), such as for amino acids, etc. Compounds provided herein that are drawn or described as having certain stereoisomeric configurations include only the indicated compounds. Compounds provided herein that are drawn or described with undefined stereochemistry include all such possible isomers, including their racemic and optically pure forms. All tautomeric forms of the compounds provided herein are included unless otherwise indicated.

[0220] The compounds described herein include variations in which one or more atoms are replaced with a non-radioactive isotope or radioactive isotope of the indicated element. For example, compounds herein that comprise hydrogen atoms encompass all possible deuterium substitutions for each of the .sup.1H hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include but are not limited to: .sup.2H or .sup.3H in place of .sup.1H, .sup.13C or .sup.14C in place of .sup.12C, .sup.15N in place of .sup.14N, .sup.17O or .sup.18O in place of .sup.16O, and .sup.33S, .sup.34S, .sup.35S, or .sup.36S in place of .sup.32S. In certain embodiments, non-radioactive isotopic substitutions may impart new properties on the oligomeric compound that are beneficial for use as a therapeutic or research tool. In certain embodiments, radioactive isotopic substitutions may make the compound suitable for research or diagnostic purposes such as imaging.

Example 1: Localization of Modified Oligonucleotides in Cells Expressing FUS

Compounds

[0221] Compounds comprising modified oligonucleotides were prepared using standard oligonucleotide synthesis techniques well known in the art. The compounds in the table below comprise modified oligonucleotides that are 5-10-5 cEt gapmers, wherein the central gap segment contains ten 2'-deoxynucleosides and is flanked by wing segments on the 3' and 5' ends, each containing five bicyclic nucleosides with a cEt (2',4'-constrained ethyl) modification. Every internucleoside linkage of each oligonucleotide is a phosphorothioate (PS) linkage. The nucleobase sequences of the modified oligonucleotides are either 100% complementary to the genomic sequence of human PTEN (GENBANK No. NM_030059.12, truncated from 8370000 to 8482000, herein referred to as SEQ ID No. 1) or are not 100% complementary to any known human gene. The compounds in the table below also comprise a Cy3 or Alex Fluor 594 conjugate group in order to allow detection of the oligonucleotides in cells.

TABLE-US-00001 TABLE 1 Compounds comprising modified oligonucleotides SEQ Compound 5' End ID ID Cap Sequence Target No. 598987 Cy3 .sup.mC.sub.ksT.sub.ksG.sub.ks.sup.mC.sub.ksT.sub.ksA.sub.dsG.s- ub.ds.sup.mC.sub.ds.sup.mC.sub.dsT.sub.ds PTEN 2 .sup.mC.sub.dsT.sub.dsG.sub.dsG.sub.dsA.sub.dsT.sub.ksT.sub.ksT.sub.ksG.- sub.ksA.sub.k 766635 AF594 .sup.mC.sub.ksT.sub.ksG.sub.ks.sup.mC.sub.ksT.sub.ksA.sub.dsG.sub.ds.sup.- mC.sub.ds.sup.mC.sub.dsT.sub.ds PTEN 2 .sup.mC.sub.dsT.sub.dsG.sub.dsG.sub.dsA.sub.dsT.sub.ksT.sub.ksT.sub.ksG.- sub.ksA.sub.k 950431 Cy3 .sup.mC.sub.ks.sup.mC.sub.ksT.sub.ksT.sub.ks.sup.mC.sub.ks.sup.- mC.sub.ds.sup.mC.sub.dsT.sub.dsG.sub.dsA.sub.ds none 3 A.sub.dsG.sub.dsG.sub.dsT.sub.dsT.sub.ds.sup.mC.sub.ks.sup.mC.sub.ksT.su- b.ks.sup.mC.sub.ks.sup.mC.sub.k Subscripts: "s" indicates a phosphorothioate internucleoside linkage; "k" indicates a 2',4'-constrained ethyl bicyclic sugar moiety (cEt); "d" indicates a 2'-deoxyribo unmodified sugar moiety. Superscript m preceding a "C" indicates a 5-methylcytosine.

Experimental Protocol

[0222] HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin and seeded at 12,500 cells/cm.sup.2 on collagen-coated coverslips (for immunofluorescence detection of 598987) or 35 mm collagen-coated live imaging dishes (P35GCOL-1.5-14-C, MatTek, Ashland, Mass.) (for live cell imaging). Plasmids containing a pCMV promoter and either tGFP-FUS(WT) or tGFP-FUS(P525L) were individually mixed with the TurboFect transfection reagent (Thermo Fisher Scientific) in Opti-MEM and incubated for 15 min at room temperature. HeLa cells were then treated for 16-24 hours per manufacturer's instructions for transient transfection. Cells were then washed once with PBS and incubated for 4-6 hours in Opti-MEM containing a final concentration of 50 nM of a compound listed in the table above. In a separate experiment, the cells were incubated with 50 nM with compound 598987 first, washed, and then transiently transfected with tGFP-FUS-P525L as above.

[0223] Confocal microscopy was used to visualize the cells. Confocal images were acquired on an Olympus FV1000 microscope using a PlanApo N 60.times.O objective (N.A.=1.42) with excitation laser lines at 450, 488, 542, and 635 nm. For immunofluorescence imaging, cells were fixed with 4% formaldehyde in PBS for 30 minutes at room temperature, permeabilized for 5 minutes with 0.1% Triton-X 100, washed three times with PBS, and blocked for 30 minutes at room temperature with blocking buffer (1 mg/mL BSA in PBS). Primary antibody incubation for G3BP protein (mouse-anti-G3BP, Abcam ab56574, 1:600) was performed for 2 hours at room temperature or overnight at 4.degree. C. in blocking buffer, followed by 3 washes of 0.1% Nonidet P40 substitute 74385 (Sigma-Aldrich) in PBS. Secondary antibody (goat anti-mouse IgG (H+L)-Alexa Fluor 488, Jackson ImmunoResearch 115-525-146) was incubated 1:200 in blocking buffer for 1 hour at room temperature, followed by 3 washes.

[0224] Co-localization analysis of compounds with G3BP was performed using the JACoP plugin for ImagJ-Fiji using images captured under identical non-saturating exposure settings (Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B. et al. (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods, 9, 676-682; Bolte, S. and Cordelieres, F. P. (2006) A guided tour into subcellular co-localization analysis in light microscopy. J Microsc, 224, 213-232). The thresholded Manders' co-localization coefficient was calculated using constant maximum and minimum threshold values within a set of conditions to be compared. Values reported in a single table were obtained using the same image exposure settings and threshold values. Inverted co-localization is a control value obtained by rotating one of the two images being compared 90 degrees and performing the same analysis. See Bolte, 2006 for a more in-depth discussion of JACoP software and the threshold Manders' coefficient. For image co-localization analysis, each field contained an average of approximately four cells. A high co-localization coefficient indicates co-localization with the G3BP stress granule marker, while a lower co-localization coefficient indicates random distribution relative to G3BP.

[0225] As an additional semi-quantitative way to analyze localization of compounds comprising oligonucleotides in cells, comparative quantification of granule/nuclear intensity was done by image analysis using ImageJ-FIJI macro scripts. First, images were captured under identical non-saturating exposure settings, and then the average pixel intensity in the nucleus was calculated based on the DAPI channel. An absolute intensity threshold was used to create a nuclear selection mask. In the Cy3 channel, a background subtraction was performed and the average pixel intensity in the nuclear selection was measured. For quantification of average pixel intensity in the tGFP channel (tGFP, tGFP-FUS-P525L, tGFP-PSF-.DELTA.NLS channels), a uniform absolute intensity threshold was applied to create a tGFP (granule) selection mask. In the Cy3 channel, a background subtraction was performed in this selection and the average pixel intensity in the tGFP selection was measured. Results are presented as the average ratio of granule/nuclear intensity for 12-18 cells for each condition.

[0226] For live cell imaging, cells were treated with 1 .mu.g/mL Hoechst 33342 (Thermo Fisher) and imaged in FluoroBrite DMEM (Thermo Fisher) at 37.degree. C.

Imaging Results

Fixed Cells

[0227] In fixed HeLa cells transiently transfected with tGFP-FUS-WT, a diffuse GFP signal localized to the nucleus of cells, overlapping with the signal from the nuclear stain DAPI, and no GFP signal was observed in the cytoplasm. In cells transiently transfected with tGFP-FUS-P525L, the GFP signal was instead observed as bright spots in the cytoplasm, non-overlapping with nuclear DAPI stain. These cytoplasmic spots overlapped with the G3BP stress granule maker.

[0228] When cells expressing tGFP-FUS-WT were treated with compound 598987, the Cy3 signal was visible throughout the image, and was more prevalent in the nucleus than the cytoplasm. In contrast, when cells expressing tGFP-FUS-P525L were treated with 598987, the Cy3 signal localizes to the G3BP-containing granules in the cytoplasm. This result indicated that the P525L mutation of FUS caused localization of FUS to change from the nucleus to the cytoplasm and co-localization with G3BP-positive granules. The localization of the cEt modified oligonucleotide also changed from primarily in the nucleus to the cytoplasm where it also co-localized with granules containing the mutant FUS.

[0229] A similar result was observed with two other cEt modified oligonucleotides. In fixed HeLa cells transiently transfected with tGFP-FUS-WT and then treated with compound 950431 or 766635, no co-localization was seen between the compound and G3BP. In contrast, statistically significant (p<0.001) co-localization was observed between the compound and G3BP in cells transiently transfected with tGFP-FUS-P525L and then treated with compound 950431 or 766635.

TABLE-US-00002 TABLE 2 Thresholded Mander's co-localization coefficient: compound with G3BP (%) Expressed Colocalization Inverted Colocalization Compound Protein Coeffcient Coefficient 766635 tGFP 11.5 9.6 tGFP-FUS-WT 11.6 2.8 tGFP-FUS-P525L 30.2 7.3

TABLE-US-00003 TABLE 3 Thresholded Mander's co-localization coefficient: compound with G3BP (%) Expressed Colocalization Inverted Colocalization Compound Protein Coeffcient Coefficient 950431 tGFP 9.1 6.8 tGFP-FUS-WT 4.1 2.2 tGFP-FUS-P525L 33.9 5.6

TABLE-US-00004 TABLE 4 Granule/Nuclear Ratio Compound Expressed Protein Granule/Nuclear Ratio 766635 tGFP 0.55 tGFP-FUS-WT 1.00 tGFP-FUS-P525L 3.88

TABLE-US-00005 TABLE 5 Granule/Nuclear Ratio Compound Expressed Protein Granule/Nuclear Ratio 950431 tGFP 0.58 tGFP-FUS-WT 0.99 tGFP-FUS-P525L 3.03

Live Cells

[0230] Live HeLa cells were transiently transfected with either tGFP-FUS-WT or tGFP-FUS-P525L, then treated with 598987 as above. In a parallel experiment, live HeLa cells were treated with 598987 for 5 hours prior to transient transfection with tGFP-FUS-WT or tGFP-FUS-P525L, followed by live cell imaging. Granule/nuclear ratios for compound localization were determined as above.

TABLE-US-00006 TABLE 6 Granule/Nuclear Ratio Expressed Granule/ Compound, time of addition Protein Nuclear Ratio 598987, added 16 hr after tGFP-FUS-WT 0.98 plasmid transfection tGFP-FUS-P525L 1.56 598987, added before tGFP-FUS-WT 1.01 plasmid transfection tGFP-FUS-P525L 1.76

A431 Cells

[0231] To confirm that these results were not specific to the HeLa cell type, A431 cells were stably transduced with lentiviral particles (MOI .about.5) containing tGFP, tGFP-FUS-WT, or tGFP-FUS-P525L. Localization of tGFP-FUS-WT was similar to that described in HeLa cells above. In many cells, tGFP-FUS-P525L was diffuse through the cytoplasm, while in a subset of these cells, cytoplasmic aggregates were observed. A431 cells were transfected with 50 nM of compound 950431 for 5 hours. In cells with cytoplasmic aggregates of tGFP-FUS-P525L, compound 950431 co-localized with these aggregates. In cells expressing tGFP-FUS-WT, compound 950431 primarily localized to the nucleus, as observed in HeLa cells.

Example 2: Localization of Modified Oligonucleotides in Cells Expressing PSF

Background

[0232] The C-terminal nuclear localization sequence of PSF is required for nuclear localization of the protein. A mutant lacking the final 6 amino acids of the protein lacks this sequence and is defective for nuclear uptake.

Experimental Protocol

[0233] HeLa cells were transiently transfected with EGFP-PSF-WT(1-707) or EGFP-PSF-.DELTA.NLS(1-701) and treated with 50 nM of compound as described in Example 1. Confocal immunofluorescence imaging was used to visualize GFP (PSF), Cy3 (modified oligonucleotide), G3BP (cytoplasmic granule marker), and DAPI (nuclear stain), with cell fixing and labeling as described in Example 1.

Imaging Results

TABLE-US-00007

[0234] TABLE 7 Thresholded Mander's co-localization coefficient: compound with G3BP (%) Inverted Colocalization Colocalization Compound Expressed Protein Coeffcient Coefficient 766635 EGFP 11.5 9.6 EGFP-PSF-WT(1-707) 3.2 3.0 EGFP-PSF-.DELTA.NLS(1-701) 36.6 6.6

TABLE-US-00008 TABLE 8 Thresholded Mander's co-localization coefficient: compound with G3BP (%) Co- Inverted Co- localization localization Compound Expressed Protein Coeffcient Coefficient 950431 EGFP 9.1 6.8 EGFP-PSF-WT(1-707) 3.1 3.6 EGFP-PSF-.DELTA.NLS(1-701) 32.2 5.2

TABLE-US-00009 TABLE 9 Granule/Nuclear Ratio Compound Expressed Protein Granule/Nuclear Ratio 766635 tGFP 0.55 tGFP-PSF-WT(1-707) 1.00 tGFP-PSF-.DELTA.NLS(1-701) 2.65

TABLE-US-00010 TABLE 10 Granule/Nuclear Ratio Compound Expressed Protein Granule/Nuclear Ratio 950431 tGFP 0.58 tGFP-PSF-WT(1-707) 0.99 tGFP-PSF-.DELTA.NLS(1-701) 2.34

Example 3: Effect of Modified Sugar Moieties on Oligonucleotide Localization

Compounds

[0235] Compounds comprising modified oligonucleotides were prepared using standard oligonucleotide synthesis well known in the art. Compounds 446654, 598987, 626825, and 851810 are 5-10-5 gapmers, wherein each central gap segment containing ten 2'-deoxynucleosides is flanked by wing segments on the 3' and 5' ends, each containing 5 nucleosides with a modification indicated in the table below. The modified oligonucleotide of compound XL198 contains only 2'-deoxyribonucleosides. These oligonucleotides comprise full phosphothioate (full PS) linkages. The modified oligonucleotides are 100% complementary to the genomic sequence of PTEN, GENBANK No. NM_030059.12, truncated from 8370000 to 8482000, SEQ ID No. 1.

TABLE-US-00011 TABLE 11 Compounds comprising modified oligonucleotides Seq Compound 5'-End ID ID Cap Chemistry Notation Target No 446654 Cy3 .sup.mC.sub.esT.sub.esG.sub.es.sup.mC.sub.esT.sub.esA.sub.dsG.s- ub.ds.sup.mC.sub.ds.sup.mC.sub.dsT.sub.ds PTEN 2 .sup.mC.sub.dsT.sub.dsG.sub.dsG.sub.dsA.sub.dsT.sub.esT.sub.esT.sub.esG.- sub.esA.sub.e 598987 Cy3 .sup.mC.sub.ksT.sub.ksG.sub.ks.sup.mC.sub.ksT.sub.ksA.sub.dsG.s- ub.ds.sup.mC.sub.ds.sup.mC.sub.ds PTEN 2 T.sub.ds.sup.mC.sub.dsT.sub.dsG.sub.dsG.sub.dsA.sub.dsT.sub.ksT.sub.ksT.- sub.ksG.sub.ksA.sub.k 626825 Cy3 C.sub.fsU.sub.fsG.sub.fsC.sub.fsU.sub.fsA.sub.dsG.sub.ds.sup.mC- .sub.ds.sup.mC.sub.dsT.sub.ds PTEN 4 .sup.mC.sub.dsT.sub.dsG.sub.dsG.sub.dsA.sub.dsU.sub.fsU.sub.fsU.sub.fsG.- sub.fsA.sub.f XL198 Cy3 C.sub.dsT.sub.dsG.sub.dsC.sub.dsT.sub.dsA.sub.dsG.sub.dsC.sub.ds- C.sub.ds PTEN 2 T.sub.dsC.sub.dsT.sub.dsG.sub.dsG.sub.dsA.sub.dsT.sub.dsT.sub.dsT.sub.ds- G.sub.dsA.sub.d 851810 AF647 .sup.mC.sub.esT.sub.esG.sub.es.sup.mC.sub.esT.sub.esA.sub.dsG.sub.ds.sup.- mC.sub.ds.sup.mC.sub.ds PTEN 2 T.sub.ds.sup.mC.sub.dsT.sub.dsG.sub.dsG.sub.dsA.sub.dsT.sub.esT.sub.esT.- sub.esG.sub.esA.sub.e Subscripts: "s" indicates a phosphorothioate internucleoside linkage; "k" indicates a 2',4'-constrained ethyl bicyclic sugar moiety (cEt); "d" indicates a 2'-deoxyribo unmodified sugar moiety; "e" indicates a 2'-MOE sugar moiety; "f" indicates a 2'-F sugar moiety. Superscript m preceding a "C" indicates a 5-methylcytosine.

Experimental Protocol

[0236] HeLa cells were transfected with tGFP-FUS-P525L and 50 nM Cy3-modified oligonucleotide as described in Example 1 as well as 50 nM Alexa-647-labeled modified oligonucleotide, compound 851810 as a reference standard. To allow semi-quantitative comparisons among experimental groups, comparative quantification was done by image analysis using ImageJ-FIJI macro scripts. First, images were captured under identical non-saturating exposure settings, and then the average pixel intensity in the nucleus was calculated based on the DAPI channel. An absolute intensity threshold was used to create a nuclear selection mask. In the Cy3 channel, a background subtraction was performed and the average pixel intensity in the nuclear selection was measured. For quantification of average pixel intensity in the tGFP channel (tGFP, tGFP-FUS-P525L, tGFP-PSF-ANLS channels), a uniform absolute intensity threshold was applied to create a tGFP-selection mask. In the Cy3 channel, a background subtraction was performed in this selection and the average pixel intensity in the tGFP selection was measured. Results are presented as the average ratio of granule/nuclear intensity for 15-18 cells for each condition. The results indicate that the compounds comprising 2'-MOE and cEt modifications localized to cytoplasmic granules over the nucleus to a greater extent than the other compounds tested.

Results

TABLE-US-00012

[0237] TABLE 12 Granule/nuclear average pixel intensity ratio Compound Granule/nuclear ID ratio 446654 1.12 598987 2.80 626825 0.95 XL198 0.90

Example 4: Protein-Oligonucleotide Interactions

Compounds

[0238] Compounds comprising oligonucleotides were prepared using standard oligonucleotide synthesis well known in the art and are shown in the table below.

TABLE-US-00013 TABLE 13 Compounds comprising modified oligonucleotides Seq Compound 5'-End ID ID Cap Chemistry Notation Target No 766633 AF594 .sup.mC.sub.esT.sub.esG.sub.es.sup.mC.sub.esT.sub.esA.sub.dsG.sub.ds.sup.- mC.sub.ds.sup.mC.sub.ds PTEN 2 T.sub.ds.sup.mC.sub.dsT.sub.dsG.sub.dsG.sub.dsA.sub.dsT.sub.esT.sub.esT.- sub.esG.sub.esA.sub.e 766635 AF594 .sup.mC.sub.ksT.sub.ksG.sub.ks.sup.mC.sub.ksT.sub.ksA.sub.dsG.sub.ds.sup.- mC.sub.ds.sup.mC.sub.dsT.sub.ds PTEN 2 .sup.mC.sub.dsT.sub.dsG.sub.dsG.sub.dsA.sub.dsT.sub.ksT.sub.ksT.sub.ksG.- sub.ksA.sub.k 766637 AF594 C.sub.fsU.sub.fsG.sub.fsC.sub.fsU.sub.fsA.sub.dsG.sub.ds.sup.mC.sub.ds.su- p.mC.sub.dsT.sub.ds PTEN 4 .sup.mC.sub.dsT.sub.dsG.sub.dsG.sub.dsA.sub.dsU.sub.fsU.sub.fsU.sub.fsG.- sub.fsA.sub.f JB39 AF594 C.sub.roU.sub.roG.sub.roC.sub.roU.sub.roA.sub.roG.sub.roC.sub.r- oC.sub.roU.sub.roC.sub.ro PTEN 5 U.sub.roG.sub.roG.sub.roA.sub.roU.sub.roU.sub.roU.sub.roG.sub.roA.sub.r Subscripts: "s" indicates phosphorothioate internucleoside linkage; "o" indicates phosphodiester internucleoside linkage; "k" indicates 2'-4' constrained ethyl bicyclic sugar moity (cEt); "e" indicate 2'-MOE sugar moiety; "f" indicate 2'-fluoro sugar moiety; "r" indicate unmodified ribosyl sugar moiety; and "d" indicate unmodified 2'-deoxyribosyl sugar moiety. ".sup.mC" indicates 5-methylcytosine.

Proteins

[0239] DNA constructs described herein were cloned into NEB-IVT (New England Biolabs DHFR Control Plasmid) using standard protocols and expressed using the PURExpress in vitro Protein Synthesis Kit (New England Biolabs). All constructs included nanoluciferase (NLUC) and an HA tag or a FLAG tag. Aside from the control protein NLUC-HA, all constructs included all or part of FUS, as indicated by amino acid numbers in the table below. For several constructs, arginine to serine ("R/S") mutations were made for all arginine residues in one of the two RGG domains of FUS, as indicated in the table below.

Experimental Protocol

[0240] NanoBRET (bioluminescence resonance energy transfer) binding assays were performed with protein bound to magnetic beads (Vickers and Crooke. PLOS One, 11 (8), (2016).). First, the relative amount of purified protein per volume of bead suspension (based on nluc activity) was determined in 2.times. binding buffer (200 mM potassium acetate, 40 mM Tris pH 8.0, 2 mM EDTA, 0.02% NP-40, 6 .mu.g/mL BSA, and 1:1000 Promega Nano-Glo luciferase substrate) using an eight-point dilution curve over .about.3.5 orders of magnitude. The Alexafluor594-labeled modified oligonucleotides were diluted into water in opaque white 96-well plates at concentrations ranging from pM to low .mu.M in 50 .mu.L final volume. 50 .mu.L/well of 2.times. binding buffer containing 10.sup.6 RLU (relative luminescence units) beads/well was added and plates were shaken for 10 minutes at room temperature. Nanoluciferase activity and BRET were measured in a Glowmax Discover plate reader and K.sub.D values, shown in the tables below, were calculated using GraphPad Prism. The results indicate that the compound comprising 2'-F modifications bound with higher affinity to the FUS domains and mutants tested, including the low complexity domain (amino acids 1-283), than the other compounds tested.

TABLE-US-00014 TABLE 14 K.sub.D values (nM) determined using nanoBRET Compound ID Protein Construct 766633 766635 766637 NLUC-HA >1,000 >1,000 >1,000 FUS(1-283)-NLUC-HA 44.7 35.2 7.1 FUS(1-375)-NLUC-HA 16.7 12.1 1.4 FUS(284-375)-NLUC-HA >1,000 >1,000 340.4 FUS(375-526)[P525L]-NLUC-HA 36.8 30.7 4.4 FUS(1-526)[P525L]-NLUC-HA 16.4 17.2 2.2 NLUC-FUS(1-526)[P525L]-HA 16.6 12.4 1.9

TABLE-US-00015 TABLE 15 K.sub.D values (nM) determined using nanoBRET Compound ID Protein Construct 766633 766635 766637 FUS(1-375)-NLUC-HA 11.8 11.3 0.8 FUS(284-375)-NLUC-HA >1,000 >1,000 424.1 FUS(213-375)-NLUC-HA 10.0 9.0 0.9 FUS(242-375)-NLUC-HA 102.8 117.2 11.0

TABLE-US-00016 TABLE 16 K.sub.D values (nM) determined using nanoBRET Compound ID Protein Construct 766633 766635 766637 FUS(375-526)1[P252L]-NLUC-HA 23.7 19 2.9 FUS(375-421)-NLUC-HA 290.6 203 40.8 FUS(375-526)[P525L][R/S in 454- 282.3 290.1 38.4 526]-NLUC-HA FUS(375-526)[P525L][R/S in 375- 12.4 14.3 1.8 422]-NLUC-HA FUS(455-526)[P525L]-NLUC-HA 39.4 34.3 6.6

TABLE-US-00017 TABLE 17 K.sub.D values (nM) determined using nanoBRET Compound Protein Construct 766633 766635 766637 JB39 FUS(1-526)[P525L]-NLUC-FLAG 1.2 3 0.4 143.3 FUS(1-421)-FUS(455-526)[P525L]- 4.2 6.8 1.1 205.9 NLUC-FLAG FUS(1-526)[P525L][R/S in 375-422 1.6 3.7 0.3 248.2 and 454-526]-NLUC-FLAG

Example 5: Protein-Oligonucleotide Interactions in Presence of Unlabed Competitor Olignucleotide

[0241] Compounds comprising or consisting of oligonucleotides were prepared using standard oligonucleotide synthesis well known in the art and are shown in the table below.

TABLE-US-00018 TABLE 18 Compounds comprising oligonucleotides Seq Compound 5'-End ID ID Cap Chemistry Notation Target No JB39 AF594 C.sub.roU.sub.roG.sub.roC.sub.roU.sub.roA.sub.roG.sub.roC.sub.r- oC.sub.roU.sub.ro PTEN 5 C.sub.roU.sub.roG.sub.roG.sub.roA.sub.roU.sub.roU.sub.roU.sub.roG.sub.ro- A.sub.r JB40 none C.sub.doT.sub.doG.sub.doC.sub.doT.sub.doA.sub.doG.sub.doC.sub.do- C.sub.doT.sub.do PTEN 2 C.sub.doT.sub.doG.sub.doG.sub.doA.sub.doT.sub.doT.sub.doT.sub.doG.sub.do- A.sub.d B41 none C.sub.dsT.sub.dsG.sub.dsC.sub.dsT.sub.dsA.sub.dsG.sub.dsC.sub.dsC- .sub.dsT.sub.dsC.sub.ds PTEN 2 T.sub.dsG.sub.dsG.sub.dsA.sub.dsT.sub.dsT.sub.dsT.sub.dsG.sub.dsA.sub.d JB42 none C.sub.rsU.sub.rsG.sub.rsC.sub.rsU.sub.rsA.sub.rsG.sub.rsC.sub.rs- C.sub.rsU.sub.rsC.sub.rs PTEN 5 U.sub.rsG.sub.rsG.sub.rsA.sub.rsU.sub.rsUrsUrsGrsAr Subscripts: "s" indicates phosphorothioate internucleoside linkage; "o" indicates phosphodiester internucleoside linkage; "r" indicate unmodified ribosyl sugar moiety; and "d" indicate unmodified 2'-deoxyribosyl sugar moiety.

Proteins

[0242] Binding assays were performed using full length FUS[P525L]-NLUC-FLAG as described in Example 4.

Experimental Protocol

[0243] Competitive NanoBRET binding assays were performed with unlabeled oligonucleotides JB40, JB41, and JB42. First, the relative amount of purified protein per volume of bead suspension (based on nluc activity) was determined in 2.times. binding buffer (200 mM potassium acetate, 40 mM Tris pH 8.0, 2 mM EDTA, 0.02% NP-40, 6 .mu.g/mL BSA, and 1:1000 Promega Nano-Glo luciferase substrate) using an eight-point dilution curve over .about.3.5 orders of magnitude. Alexafluor594-labeled JB39 was diluted into water in opaque white 96-well plates a single concentration, and varying concentrations of JB40, JB41, or JB42, spanning the pM to low .mu.M range were added to a final total volume of 50 .mu.L/well. 50 .mu.L/well of 2.times. binding buffer containing 10.sup.6 RLU (relative luminescence units) beads/well was added and plates were shaken for 10 minutes at room temperature. Nanoluciferase activity and BRET were measured in a Glowmax Discover plate reader and K.sub.D values, shown in the table below, were calculated using GraphPad Prism. The K.sub.D values shown in the table below represent the concentration of competitor oligonucleotide required to cause 50% dissociation of the BRET pair. The results indicate that the phosphorothioate containing oligonucleotides bound the FUS mutant with higher affinity than the phosphodiester containing oligonucleotides.

TABLE-US-00019 TABLE 19 K.sub.D (nM) values determined using competitive nanoBRET Compound ID BRET pair JB40 JB41 JB42 FUS(1-526)[P525L]-NLUC-HA/ >1000 0.4 7.2 JB39

Example 6: Protein-Oligonucleotide Interactions

[0244] NanoBRET binding assays were performed as described in Example 4 using oligonucleotides described in Example 4 and .beta.23 containing protein constructs. .beta.23 is an artificially designed .beta.-sheet forming protein that aggregates in cells and that can be targeted to the cytoplasm by including a nuclear export sequence, together referred to as NES-.beta.23. Three fusion proteins containing NES-.beta.23 were constructed and used in NanoBRET binding assays: control HA-NES-.beta.23-NLUC, HA-NES-.beta.23-NLUC-FUS(375-526)[P525L], and HA-NES-.beta.23-NLUC-FUS(375-526)[P525L][R/S in 375-422 and 454-526]. The resulting K.sub.D values are shown in the table below. These protein constructs were also tested in immunofluoresence imaging experiments in HeLa cells using compounds 598987, as described in Example 1. The results in the tables below show that the compounds comprising 2'-F modifications bound the beta-sheet forming proteins with higher affinity than the other compounds tested.

TABLE-US-00020 TABLE 20 K.sub.D values (nM) determined using nanoBRET Compound ID Protein Construct 766633 766635 766637 HA-NES-.beta.23-NLUC >1,000 949.7 345.1 HA-NES-.beta.23-NLUC-FUS(375-526)[P525L] 163.7 58.4 13.8 HA-NES-.beta.23-NLUC-FUS(375-526)[P525L] >1,000 >1,000 200.1 [R/S in 375-422 and 454-526].

TABLE-US-00021 TABLE 21 Nuclear/granule intensity ratios Nuclear/granule Protein Construct intensity ratio HA-NES-.beta.23-NLUC 0.78 HA-NES-.beta.23-NLUC-FUS(375-526)[P525L] 2.15 HA-NES-.beta.23-NLUC-FUS(375-526)[P525L] 0.62 [R/S in 375-422 and 454-526].

Example 7: Protein-Oligonucleotide Interactions

[0245] Modified oligonucleotides were prepared using standard oligonucleotide synthesis well known in the art and are shown in the table below. The modified oligonucleotides are 5-10-5 gapmers, wherein the central gap segment containing ten 2'-deoxynucleosides is flanked by wing segments on the 3' and 5' ends, each containing 5 nucleosides with modified sugar moieties as indicated in the table below. These oligonucleotides comprise full phosphothioate (full PS) linkages. See table legend in Tables 1, 2, 4, and 8.

TABLE-US-00022 TABLE 22 Modified oligonucleotides Seq Compound 5'-End ID ID Cap Chemistry Notation Target No 116847 none .sup.mC.sub.esT.sub.esG.sub.es.sup.mC.sub.esT.sub.esA.sub.dsG.sub.ds.sup.- mC.sub.ds.sup.mC.sub.dsT.sub.ds PTEN 2 .sup.mC.sub.dsT.sub.dsG.sub.dsG.sub.dsA.sub.dsT.sub.esT.sub.esT.sub.esG.- sub.esA.sub.e 404130 none C.sub.fsU.sub.fsG.sub.fsC.sub.fsU.sub.fsA.sub.dsG.sub.ds.sup.mC.sub.ds.su- p.mC.sub.dsT.sub.ds PTEN 3 .sup.mC.sub.dsT.sub.dsG.sub.dsG.sub.dsA.sub.dsU.sub.fsU.sub.fsU.sub.fsG.- sub.fsA.sub.t 582801 none .sup.mC.sub.ksT.sub.ksG.sub.ks.sup.mC.sub.ksT.sub.ksA.sub.dsG.sub.ds.sup.- mC.sub.ds.sup.mC.sub.dsT.sub.ds PTEN 2 .sup.mC.sub.dsT.sub.dsG.sub.dsG.sub.dsA.sub.dsT.sub.ksT.sub.ksT.sub.ksG.- sub.ksA.sub.k

Proteins

[0246] Protein p54nrb is a nucleic acid binding protein that forms a heterodimer with PSF. HA-NLUC tagged PSF and HA-HaloTag618 (Promega) tagged p54nrb were coexpressed in HeLa cells. NLUC and HaloTag618 form a BRET pair that can be used to detect interactions between the two proteins, and BRET is observed between HA-NLUC-PSF and HA-HaloTag618-p54nrb in cell lysate.

Experimental Protocol

[0247] HeLa cells coexpressing the two proteins were lysed, and the lysate was incubated with various concentrations of modified oligonucleotide in a competition experiment. BRET signal was analyzed as in Example 4. The K.sub.D values shown in the table below represent the concentration of competitor oligonucleotide required to cause 50% dissociation of the BRET pair. The results show that the oligonucleotide comprising 2'-F modifications disrupted the p54nrb-PSF interaction more effectively than the other compounds tested.

TABLE-US-00023 TABLE 23 K.sub.D values (nM) for HA-NLUC-PSF and HA-HaloTag618-p54nrb Competitor Compound ID BRET pair 116847 582801 404130 HA-NLUC-PSF/HA- 2.46 5.12 0.66 HaloTag618-p54nrb

Example 8: Localization of Modified Oligonucleotides in Cells Induced to Produce Stress Granules

[0248] Compounds described above and in the table below were used to test oligonucleotide co-localization with induced stress granules.

TABLE-US-00024 TABLE 24 Compound comprising modified oligonucleotide Seq 5'-End ID Compound Cap Chemistry Notation Target No 391857 FITC .sup.mC.sub.lsT.sub.lsG.sub.ls.sup.mC.sub.lsT.sub.lsA.sub.dsG.sub.ds.sup.- mC.sub.ds.sup.mC.sub.dsT.sub.ds PTEN 2 .sup.mC.sub.dsT.sub.dsG.sub.dsG.sub.dsA.sub.dsT.sub.lsT.sub.lsT.sub.lsG.- sub.lsA.sub.l Subscripts: "s" indicates a phosphorothioate internucleoside linkage; "l" indicates a .beta.-D locked nucleic acid (.beta.-D LNA); "k" indicates a 2',4'-constrained ethyl bicyclic sugar moiety (cEt); "d" indicates a 2'-deoxyribo unmodified sugar moiety. Superscript m preceding a "C" indicates a 5-methylcytosine.

Experimental Protocol

[0249] HeLa cells were either transfected for 5 hours or NEON electroporated with 50 nM compound 598987 or 391857. Cells were then incubated with DMSO, sodium arsenite, or 15d-PGJ2 for 1 hour, and then imaged with confocal immunofluorescence as described in Example 1. Sodium arsenite induces stress granules through a elF2.alpha.-dependent mechanism and 15d-PGJ2 induces stress granules through a elF2.alpha.-independent mechanism. The results of co-localization of the compounds with G3BP are shown in the tables below.

Imaging Results

TABLE-US-00025

[0250] TABLE 25 Threshold Manders' Co-localization for transfected 598987 Treatment condition Co-localization Inverted co-localization Vehicle 5.9 2.5 Sodium arsenite 10.6 4.0 15d-PGJ2 11.3 2.5

TABLE-US-00026 TABLE 26 Threshold Manders' Co-localization for electroporated 598987 Treatment condition Co-localization Inverted co-localization Vehicle 1.7 1.9 Sodium arsenite 9.3 2.3 15d-PGJ2 13.0 2.0

TABLE-US-00027 TABLE 27 Threshold Manders' Co-localization for transfected 391857 Treatment condition Co-localization Inverted co-localization Vehicle 5.7 3.5 Sodium arsenite 13.5 3.0 15d-PGJ2 10.8 1.4

TABLE-US-00028 TABLE 28 Threshold Manders' Co-localization for electroporated 391857 Treatment condition Co-localization Inverted co-localization Vehicle 0.4 0.3 Sodium arsenite 5.5 1.6 15d-PGJ2 10.3 1.3

Example 9: Granule/Nuclear Ratios

[0251] Granule/nuclear ratios of compounds 598987 and 391857 in the presence of transiently transfected HA-FUS-WT or HA-FUS-P525L were measured in HeLa cells using the immunofluorescence techniques described in Example 1. HA-FUS was detected using rabbit-anti-HA (Abcam Ab9110, 1:300) with secondary antibody goat anti-rabbit IgG(H+L)-AlexaFluor488 (Jackson ImmunoResearch 111-545-155, 1:200) for 598987 and with goat anti-rabbit IgG(H+L)-Cy5 (Jackson ImmunoResearch 115-175-144, 1:200) for 391857. The results are shown in the tables below.

TABLE-US-00029 TABLE 29 Granule/Nuclear Ratio Compound Expressed Protein Granule/Nuclear Ratio 598987 HA-FUS 1.02 HA-FUS-P525L 1.57

TABLE-US-00030 TABLE 30 Granule/Nuclear Ratio Compound Expressed Protein Granule/Nuclear Ratio 391857 HA-FUS 0.99 HA-FUS-P525L 1.82

Example 10: Oligonucleotide Localization in Presence of Protein Aggregates

[0252] HA-NES-.beta.23-tGFP, HA-NES-.beta.23-tGFP-FUS(375-526)[P525L], and HA-NES-.beta.23-tGFP-FUS(375-526)[P525L] [R/S in 375-422 and 454-526] (see Example 6) were transiently transfected in HeLa cells. Cells were treated with 50 nM 598987 for 5 hr and then treated with 500 .mu.M sodium arsenite for 1 hr prior to imaging. Image analysis was performed as described in Example 1. Each value represents the average of 18-19 images.

TABLE-US-00031 TABLE 31 Granule/Nuclear Ratio for 598987 Protein Granule/nuclear ratio HA-NES-.beta.23-tGFP 0.79 HA-NES-.beta.23-tGFP-FUS(375-526)[P525L] 1.95 HA-NES-.beta.23-tGFP-FUS(375-526)[P525L] 0.75 [R/S in 375-422 and 454-526]

TABLE-US-00032 TABLE 32 Threshold Mander's co-localization coefficient of 598987 with G3BP Co- Inverted co- localization localization Protein coefficient coeffecient HA-NES-.beta.23-tGFP 12.8 4.1 HA-NES-.beta.23-tGFP-FUS(375- 7.8 1.9 526)[P525L] HA-NES-.beta.23-tGFP-FUS(375- 7.7 2.4 526)[P525L][R/S in 375-422 and 454-526]

Sequence CWU 1

1

51112001DNAHomo sapien 1ctcaccagct caggggtagt gactggacgt ttgttgcaac atcggagaat gcacgctctg 60ggctgcagca ggagataccc tcaagcacag aaccaaaagg gttcacccta agcggcaggg 120catcagcgat ggagaggccc gagagcccta gcgcccagct ccttttccca cgtttgggaa 180ggcgcagaat aggtcgatgt agagcaagga gtgagtctca ggtctcagtc ctttggcttg 240ctcttagggt agcaggcgag gagtggcacc agtttgggga ctctctcccc gcgttctgta 300agaatcggcg gcagccagca ggcggggagg cgggggcacg tgtttggatg tgggtgcttg 360tgtaaccagt tccccaagcg ccagccccga cagcgctcct tcgggaggct ggtccgagcc 420cctgtttccg ccgcggcgca ggaagggttg gggttccgct gcctgcacca ggcaagagca 480ccccgagcaa aggaagaaga cgacttgcct ccggagctat cactggggag tgggaatttg 540gaaagttccc caactaggga cacacgtgac ctccttcgga aagtagttcc gactgtggcc 600cgtgtatcct tccacctcct tttgaaccct cctaggtctc ctcgccccgc ccactcgctg 660ggctgcagct tcctaccgtt ccgtactttc cactcaaccc ggtaacccca aacgtgcacg 720gtccggccgg ggcgcgcgga gcctggcccc gggcgatcca tcctgccggg ttttcacggc 780ggccaagggg gggcggggct aggtggtctc tgagaaccga gcttgactcc gacgccgcga 840accgacctgg agcccgaggg gaaagatgct cgactctctt gggggcaccg gagcgggcgc 900aggagaggcc tgcggggtgc gtcccactca cagggatcct ctttcagttc atttagatag 960gtgccctttg ggcccttgaa attcaacggc tatgtgttca cgttcagcac gctcggctga 1020gagctttcat ttttagggca aacgagccga gttaccgggg aagcgagagg tggggcgctg 1080caagggagcc ggatgaggtg atacacgctg gcgacacaat agcaggttgc tctttgtgct 1140aagactgaca ccatgaggac acagatttgg gggaaggggg aatctctagg caaaggctgt 1200tacagtcaaa tctctgcgaa cgattgtgat ccgacagcgg tgcaaaagga aagagcgaat 1260gcagtccacg ccgcggaaat ctaggggtag aggcaagggg ggagggtatt ccccttgcag 1320ggaccgtccc tgcatttccc tctacactga gcagcgtggt cacctggtcc ttttcacctg 1380tgcacaggta acctcagact cgagtcagtg acactgctca acgcacccat ctcagctttc 1440atcatcagtc ctccaccccc gccccacaac agcctaccct gcctccggct gggtttctgg 1500gcagaggccg aggcttagct cgttatcctc gcctcgcgtt gctgcaaaag ccgcagcaag 1560tgcagctgca ggctggcggc tgggaaccgg cccgagcaag ccccaggcag ctacactggg 1620catgctcagt agagcctgcg gcttggggac tctgcgctcg cacccagagc taccgctctg 1680ccccctccta ccgccccctg ccctgccctg ccctcccctc gcccggcgcg gtcccgtccg 1740cctctcgctc gcctcccgcc tcccctcggt cttccgaggc gcccgggctc ccggcgcggc 1800ggcggagggg gcgggcaggc cggcgggcgg tgatgtggcg ggactcttta tgcgctgcgg 1860caggatacgc gctcggcgct gggacgcgac tgcgctcagt tctctcctct cggaagctgc 1920agccatgatg gaagtttgag agttgagccg ctgtgaggcg aggccgggct caggcgaggg 1980agatgagaga cggcggcggc cgcggcccgg agcccctctc agcgcctgtg agcagccgcg 2040ggggcagcgc cctcggggag ccggccggcc tgcggcggcg gcagcggcgg cgtttctcgc 2100ctcctcttcg tcttttctaa ccgtgcagcc tcttcctcgg cttctcctga aagggaaggt 2160ggaagccgtg ggctcgggcg ggagccggct gaggcgcggc ggcggcggcg gcacctcccg 2220ctcctggagc gggggggaga agcggcggcg gcggcggccg cggcggctgc agctccaggg 2280agggggtctg agtcgcctgt caccatttcc agggctggga acgccggaga gttggtctct 2340ccccttctac tgcctccaac acggcggcgg cggcggctgg cacatccagg gacccgggcc 2400ggttttaaac ctcccgtgcg ccgccgccgc accccccgtg gcccgggctc cggaggccgc 2460cggcggaggc agccgttcgg aggattattc gtcttctccc cattccgctg ccgccgctgc 2520caggcctctg gctgctgagg agaagcaggc ccagtcgctg caaccatcca gcagccgccg 2580cagcagccat tacccggctg cggtccagag ccaagcggcg gcagagcgag gggcatcagc 2640taccgccaag tccagagcca tttccatcct gcagaagaag ccccgccacc agcagcttct 2700gccatctctc tcctcctttt tcttcagcca caggctccca gacatgacag ccatcatcaa 2760agagatcgtt agcagaaaca aaaggagata tcaagaggat ggattcgact tagacttgac 2820ctgtatccat ttctgcggct gctcctcttt acctttctgt cactctctta gaacgtggga 2880gtagacggat gcgaaaatgt ccgtagtttg ggtgactata acatttaacc ctggtcaggt 2940tgctaggtca tatattttgt gtttcctttc tgtgtattca acctagggtg tgtttggcta 3000gacggaactc ttgcctggtt gcaagtgtca agccaccgat tgctttctta ggctatctat 3060atggtctctt cctgagggct attgtccgtt aatacagaat acagtacact gttagtggat 3120tagcgagctc ggtaatccgg tctcctaaat gaacaaaaaa gtagacgctt tttgaggttg 3180agcatatttc gattaaatct tggcttaggc cctagatcaa gggtttagat cagaataaaa 3240tgaaaattag tgttgcacgt acgcatattg catcagaatc ttgcagtgat tgttttagtt 3300tcctgagttg cattgataga ttcttttaaa atatgactga tttgcataac tttagaagca 3360gaatcatttt cagtatatat ggtgcacatt gagggcaaaa agtagttttg ttaatgttta 3420aacttaagtt acctacaact ttgaactgta tgtagaagtt ttgtagtttg aagtcaatag 3480tgccataata taccttataa ggcgttctta ctagatcttt gttatattta cctttttctc 3540tccctatggg gtgatgtagg atagtgcttg aaatttgcac ttcagtagca tttaatgttc 3600agtgctcttg tcataaacat agaatggata ttgagtagtt tctgatccca gatggtaatg 3660tgtaggttca agggtattgt gtgtagcaag tgaagattgc agaaataaaa cttcagttca 3720tgcttgaaat ttaagtattg ttgtgatgcc agaattgctg ctcaccgttt ttaggtttca 3780ggtcctctga caccttttgg tatcgttaat tttactgatt tgtgtagaat gtcagttgta 3840ttttaccagc taatatctag aaatgctggc aagaggggtt tactccagct ttagattgta 3900ggtatgttag cttttttcat acagtgtatt aaatttactg agtcagcttg ctgaataaga 3960cagaagccca agaattttaa cagtgtgtag ctttagttgt ctaaaagtta ggccttcggg 4020cttcaaaagt tagtggtcat cgaaaagcat taatctttgc agtttcaggt acaacacatt 4080ggttttgatt agggatgggg atggggccct ctttttgcag aatggggaaa gtattgacag 4140gaattgagag ctattggtag gccagtgtat aaggtatgtg aaaacagaat taagttattg 4200gtctgaagtg actgaagcat ttaggctcta tcaaggccta aaatttggta atatgagttt 4260ggtaatgcga attgtggcag tggacaatat ttagttaaaa ttatgtaatt gcataagtac 4320tagcacagta tttttaataa aagttatttc ttagcaaatg tcagttgcat tttgtctaaa 4380ggtagagtga cactacagtg tctatatgtc ctgctaaaaa ttgtggggaa tatttttttt 4440taagacagcg tttatatcgg gagaggtttt attccgttgg attatgttag ctgcatataa 4500atgtgcacag ttaattttgc ccaagttttt gttttgaaat gaatgtaaaa cttactgaag 4560aagtagcttc ccaaaattta gttttctgtt aagccaaaaa tattatttta aaagagtatt 4620tgcaaatttt gaagttgaca ttaattgaga atgttactaa ggctaaactg gacccgcttg 4680cccagaagat aattatggaa aaattctttt gtgacttcca aagcagtcta ctattagcat 4740gaattactga cagtcatcca aatatatagg aacaaaaaat taaatgttta tgtaactttg 4800aaaaaaaagc ctttgaagaa aataattgaa tgctgtctgg gagacagatt tctttcagca 4860cttaaagtac ataacacact acttttactt ttcccacttg attttaaatt atcagggtta 4920ttaagacctt aaaattattt taccaggttt ttacatgtga gctgtgacat gactggcatt 4980ttctttgatt tcagcgtatg ttggtctcta cacatgaaat ttgtgtgact taaaactttc 5040tctaaaactg tacttttagt tatgatatgc atagaaagca gtatcaaata ttgcgtcaaa 5100tgactaataa cacttaattt ctagagttgt ggttttattg agccaaaagt tgatatgaaa 5160aaaagtcagt aaggaaagtc agtgaagtgc ttgctttttt gataattgca ctcccaataa 5220ttttgatatt ccaacgtact tggtttgctt gttttcacgt taatgttttc tgtttattgg 5280agtgggaagg cattaaaatt gtcattgaag actttgtctt tgacatgttg tagtatttat 5340ttcagtaact aacctgtgaa aagttaaatt cctttatgaa agtagtgatt ggagtatttt 5400tatctgataa agaaagatta ataatgaagc catttctccg aggaaaattg aggacaataa 5460ttcagtttta aaatattatc gcaaaattaa attattctaa aaatttgtta gtagggttat 5520atgcttaata ttagtctgaa atatagtgct gaatttgaga ctatagaaaa attaagtgta 5580tttagggtat gtggaaacgt taggcttctg ttgtattttt attgtttggt agatttgcct 5640cttttcaaat aaatgttcac agggaatact tttaacttgt agagagtaca gtgactattg 5700aagttaccta aattacctca aggtaagtga ttactgaaat taatcatgag ggttttaaaa 5760agtattctat tcacaacatt tattttacat tgttttgtat ctgctaagtt atatttcctg 5820aaaaacatga ctggaccacc taattgctgt atgataactt acaaacttca tttttcatag 5880tgcttattca agtgtaaagc acaactgaaa ggagtaatgt acagtttata tgaggaaaaa 5940ggaattttat tgctgccagt gtaaaagttt gcacagcagt atagtcatca atgcagattt 6000acattgctta taatatacta agtaaatact aaatgattaa agataataaa atatggtgag 6060gtataaccac cttcatttta aacttagttt tagaagatag taaagaaaga ttcctttatt 6120acctttttag aattttattt ttaataacat gggaaaggca actggtgata ttttaatttt 6180tgtatggaac agtgcatctg ctttctcata gccacataaa atacataaac ttcttagtgt 6240tatgaaatgg cttacttttt ggaagtgaag aagtcttcaa ttcttatttt ctaatgttat 6300tttgaaattt gcctccattt gctgtttgtt catttggtga tagcgcaaca cttaaaaaaa 6360tattttaagc cgcttctgaa gtaatcactt cagtgacttt taatggagga gtatttgtta 6420tgggaaattc acttcacaaa gttttaacat taattcactt gaagtaaacg tgctattttt 6480aaattttcat ctcaatcttt taagtaagac gaaagcttag gaaatcactt ttattattga 6540tattgtgtgt gacttcagag tttgtaaaga gaattgtaga agtgttgcat gcatatgaca 6600attttctgct taattgaaat gtgaggcctc tgccatacta caaggattta gcttccagaa 6660aatgtaatat taacatagct taagaaatgt attttttttt ttctgtagaa accgttgggt 6720taaacaaaca gttcagaagt tttattacat ggaacccatt agttcttaat cttgttacct 6780ttttcttcat tttttctgtt aaacttgatt ttcacagtca gcattgaaga attcatcttg 6840tggcctgaat tcattaagaa aagatgttag gatttgttct gaagatagtg acttaggaaa 6900tttgtgagac tggggtcagt cagttctgtt ttacaattgc tttctatttg gtagctttga 6960aattaattta gttgcttatc agagagaata atgttgaggt tagactaacc ttaaattggt 7020aaggctttgc tgagcaaact gataactgta agtcttttat agggtgcatt actgccacat 7080atacgttctt ccataggtgg ttaaaagtat attggtctgt gtttggtgat tctctttgta 7140catattgagt atatgcattc actaatgtaa aataatttgc caagaaaggt gaaattagta 7200tattgtactt gactattcac ctttccctta gttttttgaa tttttttcat tggttgcaga 7260ggaagtatta gcaatttaat tcttttaaaa taatttgcac tggaataaat aagtatcggc 7320aaatataaga agagtaacat aatttaaggg tgaattaatt ttatttggga agttttagtt 7380ctgtatagtt aaggcagatt cttcatttgc aacagttgac attgggacat gtgtgaacat 7440cttcaaggta ttaggacatc ttcaaggcat tactttttgg cagtgttgag aatttttttt 7500tttttttttt tttttttgag atggaatctc gctctatcgc ccagtctggg gtgcagtggc 7560aggatctcgg ctcactggaa cctctgcctc ccaggttcag gcgattctcc tgcctgaacc 7620tcccaagcag ctgggattat aggtgcatgc caccacgccc agatagtttt tgtactttta 7680gtagagatgg ggttttacca ccttggccag gctggtctca aactccagac ctcaagtgat 7740ccgcctgcct cggcctccca aaatgctggg attacaggcg tgagccactg cgcctggcca 7800gtgttgagaa tattgagaga tggatattgt agctgtacct gccatcaaaa gaattttctt 7860gacctccaca tagtgtgaaa aagaagactg tttacacatt atattttaag taattataca 7920cataattatt atcagtactc accacttcaa atatgaacag tgaatctaac cagtgtttga 7980tttctctgtg tgtgtatgtg tatacaaagt tagcaaacct tttatcttaa tatttattaa 8040aaaacgaatt tttgtttctt taaagaaaag actaccttag agaatattgt tctatagttt 8100ttaaatatgg tcagatctat tttaaattat gttaaaattt tgagtattac gtttatctat 8160acttttaagc atatatacat tgtttcattt tagattttag ggaggcagtg tgggctctgg 8220agccagactg cttgttttgt aatcctggat cttccattta gtagctggat gactttgagt 8280aggttattta gattttctca atctatttta tctgtaaaat ggggatgata atggaaccta 8340ccgcatacgt ttatcttgaa tagtaagtga gataataata agtaatttca tttagcatag 8400tacctgccac attgtaaata cttaaatggt agctactgct ctgaaaaact gtaatttcag 8460gttatgtatg tagggaaatt atttgtattt tcatttatgg tgtatgattg taactgaatt 8520tcctcagttt gggccatgtt aggattttgt ttcaagttat aagtgttttt aaaaataagg 8580gtattccttt aggaagtctg ggtatgacat gtctgtgatt ttgctggttc atcacaaatg 8640ggaaataaat ctctgctaac tcaaactgtt gaccaaagta aaattaatta tgccaatcaa 8700aaactatttg ctttaaaata taaaaggcaa aaacttccta ttagcataat gaagtagaat 8760ttttaaactt tgttataatc ttaaattttc tttagtgttg aagataggtc aacttaacta 8820tcatacattt ttattcacat aaagtaaact ctgcctcaaa tgtaataaac ttaatatgag 8880ttatgtaaac tttggtcaat agaggtatat tttttagcat ttccttttga aaatttcagc 8940cttttgaggg agtcttgcaa ctgaatgtca agttacattt attacaataa aatggacact 9000taatataatc tgtaatgcat taacataata tgggaacttt taaagtattc agtctctgta 9060ttattgagtc ctatttccac atttggccag gattctcaat atgatttagg cccaagacgt 9120gggaagaaag aagtaaagaa ctaaaggatt tttttcttca tttttttaat tgaatatggg 9180gaaagatgga ataagcttat ctgtccagta aaggccatta tgtgtacata gggattatta 9240tttttccccc ccttgggctg tactgatttc ccagatgtac cacagcactc ttagtagtga 9300agcacttgac ttctagtgag tggatttttt gtgtgtgtgt tttatattgc agagtgaata 9360cactctgtct gatactatgt gactttctga ttatgtgatt tttatgcatt ttatgtgttt 9420tgtaaactag ctgtattttt ggtccatgtc taggttgtag aattgaattg tgcattttgg 9480catctgagca cagctgagtt ttctaaatca atctctctcc ttgcacctag tttttgcttt 9540agatcactac ctaagactta ctgttgattt aatattagag cacttaagca tagctttgac 9600ttttatttcc tttgattttt gtagattttc aggctgaagt acaataaggt tctctgttct 9660ttactagtaa ttgcaaagat tgtattctgt gaattttatt tgtttaatac ttttgatctt 9720ttgaagagga tgtaattatt taaggtatta tgaaatgcat tgtgatttga attagatact 9780ctttggagat ggagttttgc tgttgttgcc caggctggag tgcaatggtg tgatctcggc 9840tcaccacagc ctccgcctcc tgggttcaag caattctctt gccgcagcct cccaagtagc 9900tggaattaca ggcatgcgcc accacgcccg gctaatttta tatttttttt ttcagtagag 9960atggggggtt tctccatgtt ggtcaggctg gcctcgaact cttgacctca ggtaatctgc 10020ctgtctcagc ctgctaaagt gctgagatta caggcatgag ccactgcgcc cggcctcaga 10080tactctttta attagatgcg tttaaaaatt taacccacca ttgctggcat gaatagatgt 10140atttttagag tgattcataa atatcgtata catgtttaaa gttacaaact ttttgcttat 10200ttcaaaatgc aggattcttt tccatttaaa attccctctc tttgtgagac ttctttttga 10260gtattctggt tactctaaac tgattggaga tgaaattaga tagaattgaa aactgtactt 10320ttaaaatgaa attttgggga tgtcattaag cttgattttt taggtttttt ttttagtgtg 10380tattataaat tattttacac tgattgtcag cgataaaatg gaatgcctgg gattttttaa 10440aatttatttt attcattttt ataaggtaaa aacagtgttt tgctaggctt aatttgacca 10500tgttgtaaaa tttattgtat accttgaaag aatcatttat gaaagatact gaattagcta 10560atatatactc tgtcttatgt agtttttgat taacaataca ctttttaaat cattagctca 10620tttgattttg caaagaagaa caggtaacct aagaggcaga cagaacaggc attactttta 10680tttttctttc ttttttattt tatttattta tttatttatt tattttttgc agcttaggaa 10740ttgtagctcc agtggaatca gtatcttgtt aatggctagt gaaagactga gtctgaagaa 10800ggatgcagga cttttttggc acttggtgca gtatttttcc cattatgtta catgagtggt 10860tcttaaactt cagtgtgtta gaacaacctg aagggcttat taagctatgg attgcttact 10920ccaccctcag agtttctgat tcagtaggtc tggattggga cctgagaatt tttatttctt 10980agaagttttc aagtgatgct gatgctggtg ctctggggat cacactttga ggaccaccaa 11040tgaacattat ctcccaccaa gcaaaccctt aacatgttat actcctttag gttattagaa 11100tttatacatg cattatttca tttgacctgt aaactctaag taactttgca tggaaaatgt 11160tatcctgatt ttatagacga gatagtgagt ttagaaaggc agtatggtgg aatggagcat 11220agatttggag ttggctagac ctaaagtcca gattaaatct ctgctcaagg ctgggcgtgg 11280tggctcatgc ctgtaatccc agtgctttgg gaggccagcg ttggcagatt gcttgagtct 11340ggaagttcga gaccagtctg ggcaacatag gcagaccctg tctctacaaa aaaaaataca 11400aaaattagtc gggtgttata gtgcgcattg gtagtcccag ctactgagga ggctgaggtg 11460ggatcacctg agactgggac tttgaggctg cattgagctg tgattgggac actgtactcc 11520tgcctgggtg acagagtgag accctctctc aaaaataaat aaataaatac atccccgctc 11580agccacttat cagttacgta gatacactgc ctaaccttag tgaaccctgt ttcgacaact 11640ccaaaatggg agtaaaaatc ctaaacttgt acagtggttt tttagttttg ttaaaagtac 11700aggtgaggtt tttttcagag tattggttgc catctgagag tgatcccctt tcacctcctc 11760taggactttt agcattttct ggagacattt tggtggtcac agctggggtg gtagagtgtg 11820ctattggcta ggggcttgaa gccagtgatg ctgcttaaca tcctatatgg cacaagaccc 11880ctccccatca acaaagaatt atctagccca aaatgctgtg taaaatgtct ggtatataat 11940aagtataata tttgatgaaa atcagtacct ttgcccccag gtgtgatatt taagaaggtc 12000aacttactaa atcagtgatg gagttagtcc taacatctgg gtgttctgac tgctgctagg 12060ccagtattct ttatatgata ataagaactt tgtccacaga agatatccct aataacaaaa 12120aaggtttatt tgaagaggac tcatgtgttc tttggctgat tgtgaaagtg ttgctttgaa 12180cttctgttag aaaaggttga agatgttttc cgtaagtgtt tttaatactg tacgtagtat 12240tcagaaggat gtttaatttt ttttttaatt ttgctagtag tttttaaagt aatccttttt 12300cctttaatta tgtagttgtt gaactgttgg gagttacttt tctcttacta ttttgttatt 12360taatgtattc tttgacctta tgctttttta ttctaaagct gcttttatta tagtcagata 12420tgatgaagtt aaatgtacaa tgtaaaattg caaatttcca acgagctata caaacttaaa 12480tatttctaag taaagaaaat agggctgact ctaaggttct ttgatccatg tgttgcattc 12540ttttctaggc cctaaatttg ctatgccagc ctgttgaatt aaagtgcttt atttatctaa 12600attagaaact tgtattaaag tgaagtttta gaaaaaaaga aacaaaatcg gaatggagtt 12660ttaggttagc ccagagatgg gaagatgcca agaaggtagc tttagtggat tctgaatttt 12720ttggttttgt tttgttttta gggcaggcaa atgtaattac aaaagggttc taggaataga 12780ttgctgtgat tttttttctg tttgcatgat tttacagttt gctttgcctc tcacttttga 12840atgcagaata aaatgtcaag gccttatttt tttttaaatt cttaagaaat ttaagatttg 12900actgttaatt ccttttgaaa tatgggatat tttgagatac caattattta agacaaatag 12960gactcattgt tacaattcag ttgaataagg cttatgatgt ttatttcagt atatgaatga 13020aaactatgtg cttattgtac ttaagaaaat ttcttttatt aaaaacatga ctaaagagaa 13080ttttaaaaat cacccactgt cctacttctc taaaacttaa tgttttcata ttagcttcca 13140gttttgttca tatgcatata ctttaaaacc tagttcatgg tgaacttaag agggtgttct 13200ttttaaaaaa caatttccat tgcactttgt cgttgcctta attaaatggt gaaatcatca 13260gaaatattta ttttcctata cttatacatt tattaagctt gtttccattt ttttattttg 13320tgatttttta agtggattta agataaccta aacattagag aggattttca tggttttgat 13380tcatgaaatc ataatgttat acaaacctaa ctgaagtgtt agagccttga agatttttcc 13440cccgaattac atatagtaac tctacttgta tttaatactg aaagcatatt ttacttattt 13500aagtgagaca aagtaaaatt tagctgaata ctttagatct atcatttcct tttcctgttg 13560taagaacatt acattgtgtt gaaattaaag tggatataga aggtaattag aataaactgc 13620cacatcattt ttatagtaaa gtggtaataa cactattgct ttctgttttt ttaatcagaa 13680ggagtatggg cttataatga tgttactgtt ccctgaagca tattttgaat gatacggttt 13740atatttgcac agttgcccag gtaatcattg tgatattaat tgatcaattt gctatttatt 13800tgcgttttaa atcagtacta gtatttgtgc ttaaaaattt tgcatatgtt ttatcagatt 13860taatttttaa gtgtcagata ctaaaacaaa taaccttaac tttattaaat tataattttt 13920tatcatgagg tggtattcat ttattcatat agttagaaca aaaaatattt aaaatattga 13980ggtagaaaca aattagtctc tttttaatta aaagccagat tacttgttag agtaacattt 14040tcccaaatga ggtaaaattg ttgcgactgt taaacttaag gaaattttga tctaggtgtg 14100gtatatacct tcttgtgggg tgctaatgaa aacagggatg gcaaaaatat tttgtttgtg 14160agtgtatgca tttatgcttt ttgacaacct aagaaacact cttacatctg agtatctttc 14220atggactagc tgtaggaaat ctatataaaa tagcttagta tactgaaagt atgacatagt 14280tttacatatc tagattgtgg ttgtgattat atataatact ataaaatatg ctaacgtgct 14340gcttaataat actatttgga ttttttttaa tactgaaaag gtcacacaga ttgtgattat 14400tgtgtagtgt ccaagaacta aggcctacca tctgttactc aaatgtatga aaaagttaag 14460ataatttagt gatataagtg gttttgacac cactgttttt ggaataatct aattatgatt 14520tttataaaga ctaatatcaa attttaaacg tttgcaaaaa tgaaacctaa tagttatact 14580gttatttata tttttctatt acaatacaga tactggctga gaactaaaga ttgtgtaata 14640aacgcctggc cttcagtcat ttggtttttt ttttccctcg attgtttgga tagttaactg 14700gacatcatgt tttaacttga gaaattaagt tatacaagat tttgatattt taaactagtt 14760ttcctaactg gttgagatat ataagaattt agtattacag gactcaatca gggaactgat 14820ttaataagaa tttcttaaaa atttgtttaa atatttttca agttcttttc ttcatcttct 14880acaacttaat tcttgtctgt atgcaaatga gcttccccat ttaaaatttt gctgttgcat 14940tttaggccac tatagaagtt gtttctttaa ttttcactca caagaatttg gtcttaccaa 15000attgtgtaaa tctttaaaat tgtgtatttg gcttaatatt

atagaatctg attgatttaa 15060tctaccttgt ttcatttagt atgttgacat tttcttgaga aatttgttat gccaaatgat 15120taacataata atattttaag tttagatatg attttgaatt tacattttca aatgcaactt 15180tgtgtctgtg gccttttttt tttttttttt tttacgagaa acatcttgcc aattttcaga 15240ttaatctgtg aggaaagtag attggtttac tagactcagt ttgtagactt tggtgagaac 15300tgaattggag gctatgaaaa aaataccttt tgggcctttc tgaatagaca tatatacata 15360aattatatct cttacattaa gtgaggcaca tatgtaggtg agatttttac ctgaatatta 15420aaagtttaaa agtcgttacc tattctgttt acttaatagt atttaaaggg tgtgagaggt 15480gttatgtgtt tctgtccctt gtttttattc ctatccctcc catctaactg ttggtactct 15540tatcttccca ggtattaaac ttgtatgttt taaaagctta tttacttgtt gaaatggtta 15600acttaattag ttttttcttt gaagtttcag cctaaatatt ttctgttttt ttatatgtcc 15660tttaaatatg aaaattctac agctaatcat aattagtaat tgtacttttt cccctattac 15720aataactggt ttcataataa aatggtatcc cttcaataac aagcatttat agtagtttat 15780taaaactaag ggtgttatct attcaaacca agcaatgcag acttactgtt gactctgtta 15840atatatttta aaattgcata ttactaaaat ttaaaatatg attttgacta gtattttggt 15900gtatgttatt ttagatattt tgattatgca ctacttaaga atgaattgtc aagtatgatt 15960ataaagttga tataatagtt aaccttcagt gggaatagga atcatttaat attgttagat 16020atttgtatta ttagaacaat ctcctatgat tcttactaat atagtaatga atgacagaca 16080atatgttggc tttcatattt aaaaattcac atgcatttct agtttatgtt tttcttcgac 16140taaaattctg cagcacttag gcaaagctat ttttaccagt tggaaaaaaa gtaagtcatt 16200tccaaccaat tttcctggct tgtagtatag aataaagaga cttgatttta ttacattaaa 16260gccaaatata aaatgatgca atctagcaca cacttgtttg gaacttttct cttttaaata 16320ttcagattaa gaggacgttg aaaggtaaat tttttttttt ttttgagacg gagtctagct 16380ctgtcaccca ggctggagtg cactggcacg atttcggctt actgcaagct ctgcctccca 16440ggttcatgcc attctcctgc ctcagcctcc cgagtagctg ggactacagg cgcctaccac 16500ggtggcgccc ggctaatttt ttgtattttt agtagagacg gggtttcacc gtgttagcca 16560ggatggtctc gatctcctga cctcgtgatc tgcctgcctc ggcctcccaa agtgctggga 16620ttacaggtaa attttgattg ttattagcaa ctataaaagt tttgcagttg gcttattgga 16680aaaagaaaac ctccttgccg gagacggaga cgcatttgta ttagaacttt gttttctgag 16740taccttacct atagtaggtt tcaaatattg gtgaattagt tgatggttag gtctgcataa 16800ttactgcgta tggaaattct ggaaccctat tttttcaaaa tgcagctaat gttgagagaa 16860tatgcactaa atattactag atctttgttt ttcaagatgc tgatatccct taacatcttc 16920tgcactttac ctgtttgaat atcttttttg ctgtaaaaat tagtggcctt atgtctttct 16980gcataattat agagtagcca aaacctgttt taggttaatc acctctggca aaataaatga 17040taaaagcata gcttttgtaa gcagaatgat attacagaag ttaacttata aatctaagtg 17100tattaaagac acttaggaaa tttatgataa tgctgggtca gcattacagt tttaactttt 17160tacagttttt catatgcttt ttttgtgatt ttgctgtaga aaattaacag ttggcatttg 17220gcttagttca agtataatgc tgttgacaag tatatctgac acgtcattga actaataata 17280tttttgaaag ctgataggta agttatatct attttgtttc attcgtcatt agtgatcggt 17340cttagatgtt tttagcgaga gcaaaactgt agaggaatgt gtgtctgtgt gtgtatatgt 17400gtgtgtgtgt gtgtgtattt taacagcagg agagttctga aacaggaaac cagtcttatc 17460atattcatcc agagacctag gaagaaggta attgtttggt atactcgtta aaaccagttg 17520gttgggcaac ttaaattttt agaggatcac agatgtaggc ttgagcagtt gtagatagat 17580gatttctttt tttttctttc ttttttcttt tttttttgag atagagtctc tctctgtcat 17640ccaggctgga gtgcggtggc gcgatcttgg cttactgcaa cctctgcctc ccaggttcag 17700gcgtttctcc tgtctcagcc tcctgagtag ctgggattac aggcgcatgc tgccatgccc 17760ggctaatttt ttgtatttta gtagagacgg ggtttcaccg tgttccccag gctggtctcg 17820aactcctgag ctcaggcaat ctacccacct cggcctccca aagtgctggg attacaggcg 17880tgagccaccg tgtctggcga tagattattt cataattaac acctgctatg aagaaaaatt 17940gattaaaata gttgagaagt ctagtacact ctcagctaat atactaaatt atactatgga 18000ttttagagta ttgttaacat tatcagtgac ttgatatctt cctgaggttc taatttgctt 18060aactttaaat aattggggtt cagatcacct tgattgttcc cttaaagatt aaattttgta 18120aaactgtgtg taattttcct gtatctggtt tggatagctt taaaaatggt tcttaagttt 18180aatgagttca actgggaaaa aagttagttc tattttagat gttgtgtcac tggaaattat 18240gtttccctgt ttgttatatg cacattatta caaagttgta atcaatgttt tcatactgtt 18300ctctggtctg tttttttcac aaatacactt tttatttgtc gccaggtact tatttttaaa 18360gctatagagg taatatttca tcaggtgagg gtaactacca tggtttgttt gctatactgt 18420gttagggtta ttttcgtttt ttttttcttt tataaactat agttgtgaat atgtttatgt 18480agtttacttt tggtttatta gaatatattg ctagagtggg attacaggat taaagagtgt 18540acagtatttt agtttttttt ttttttttac aagttgcaga tttgttgcca aatgaacgag 18600tttgtagtat tgctaacaag gagaagaatt actagcaagt cttgatgtta cttttgaaga 18660gtgtgatgat tgcatttagg aagatatcta aacttctgtt tcaaagcaaa aagtatgtgc 18720aaatttctta ctcatgacaa attcatataa tataaaaaca tgaaagttgt gaggtcaggt 18780tgtttggaga agtagaaaac ttcagtagag tttatagata ggcagtcttc ctttctggtt 18840tggcactgac agcagattaa ctagaaagtg ttagaaggaa cctaaaattt atactaaagt 18900caatttaagt taattaatat accagaattc cttcttttac aatttattta taaaaacacc 18960atattgagtt gccttgtaat gagacattta aactaaattt aaataacaga attcatgcac 19020catctaataa caacccctta tttacaatta tagagtcctt tgcaatttta tagatatttt 19080catgtatccc atttggtcct tgaaacaatt aatgaagaag ttacagcaag tggtattatc 19140attattttac agaagaacaa aacaaaaata tatgtggccc agagattgag tgatttacct 19200gaggttatag gctttagatt gcatagctgg aagtagaacc ttgttcttct atattaaatg 19260acaatattca ttaagtactt agcacaggat ttggtaccta gtaaatattt aaatgttcct 19320gtgttattcc tgactattcc ttctttattc ttaaaacgcc attttttgag cactcttaat 19380atttatagtt caaactttgt acctatgtac ctttttctct ttagaaaata agatttcagg 19440ctgcattaat ttgatctgta caggaatgat tatatgtttt acatattggg acaaattgct 19500ctttttttat ataccttaag ctctagggta catgtgcaca acatacagat ttgttacata 19560tgtatacatg tgccatgtta gtgtgctgca cccattaact catcacttac attaggtata 19620tctcctaatg ctatccctcc cccctcccca taccccatga caggccctgg tgtgtgatgt 19680tccccaccct gtgtccaagt gttctcattg ttcagttccc acctatgagt gagaacacgc 19740ggtgtttggt tttctgtcct tgcgatagtt tgctcagaat gatggtttct agcttcatcc 19800atgtccctac caaggacatg aagctcatcc ttttttatgg ctgcatagta ttccatggtg 19860tctatgtgtc acattttctt aatccagtct atcattgatg gacatttggg ttggttccaa 19920gtctttgcta ttgtgaatag tgctgcagta aacatacatg tgcatgtgtc tttatagcag 19980catgatttat actcctttgg gtatataccc agtaatggga ttgctgggtc aaatggtatt 20040tctagttcta gatcactgag gaattgccac actgacttga actagtttac agtcccacca 20100acagtgtaaa agtgttcctg tttctccaca tcctctccag cacctgttgt ttcctgactt 20160tttaatgatc actattctaa ctggtgtgag atggtatctc attgtggttt tgatttgcat 20220ttctctgatg gccagtgatg atgagcattt tttcatgtgt ctgttcgctg cataaatgta 20280ttcttttgag tagtgtctgt tcatatcctt cgcccacttt ttgatggggt tgtttgattt 20340tttcttggaa gtttgtttaa gttctttgta gattctggat attagccctt tgtcagatgg 20400gtagattgta aaagttttct cccattctct aggttgcctg ttcactctca tggtagtttc 20460ttttgctgtg cagaagctct ttaggacaaa ttgttcttaa ataatgaaca gttggcactt 20520tttcaactgg aaaattcaag gaactgctct ttctgctttc tgctcaatat gaatcttcaa 20580tttagaaatg agagtccatc attaacaatt caacatagct tattaatagg aaaaaaaaac 20640ctagtaacaa atgtaaaatc tttgattaaa tgagaaagtc atagaagttc atcagatttg 20700tatttaaagc atgatttcat tagaaaagtt gataataagg atttaactgt gacataattg 20760gaaaatactt gtttaaactt aaaattttga aaagaaatgt aaatgtgatg taacttatga 20820atcagtggtt gagtttcttt tttgctcaca agaaccctaa ctgtgtgtta cttgaaagca 20880ctgatggaaa tcagggaaaa agctccagaa gttcctacga aataaaatta aatgataaag 20940tcctggtatc tgctaacttg ccttccattc ctgttatctt ttcttcttag tctgacttca 21000ttaattcttt caccctggct actggtttag ctcagtgttt tatgagccag gcagcttcag 21060actttgcttt tgatgctctt tgttcattac ctctaaagct gtattatcac tttcatttta 21120tcattaatgt ttcatgtata tgttatagtt tcatattgtt actgcaactt ttacttagct 21180ataatttaaa aaatatctgt gatctgtgga aataattatt ctatggcaga aaagtagtta 21240ttgcatttta ctttataagt tgtttaagga taagcatacc tatatattaa gcactacaaa 21300gaaactttta caatggcttt atttttagca aaccatcata gttaaaataa gatttagtgt 21360acatgtcagg aacacagtct tatgaaataa ggtttaggga gctattttta gttactatat 21420cctacttgaa aattgtagtt aaatttctag catataccct attaatttag atgcaagtac 21480agatttgaga taaggtagat acattatttg gatgtcaact cggaagttgt tcaagaaaag 21540atattttgtt atttagatgt aacttggaac atatttctag tgtttcaagt catgattgta 21600tgcctagaac aggcaataaa aatttactta gctggtaaaa cagccacatt atttcaaata 21660tagtttagtt atattatgga ttaaattgat ttttgtggac agactttaga acttaattgc 21720tattaattac attttttctt tgggacggta tttgttcttt ggtgagaaag gattcttgta 21780acacctaaat caagactgtc caaacatggc atatgcagtt taccagtcaa aacaaactaa 21840tcacttaaga tctgtgtatt ttgttttatt tgaattatac ctcaattaaa acaaatttag 21900catgtttaca caaaggtggg gggaaactgt ctaatatatc tgagatctgt tggagctggg 21960gtgaccatcc aacttaggca agatagctag taccatgtat gcatttcttt tacctttctt 22020attgtatata cagtaatcac tgatattatg agaaaaagaa ctttttaata attcaggaga 22080tattttatcg ctagtatata ttgagtgctt attatgtgcc ttatatactt tggactcatt 22140taatcctcaa cacaacaacc ctatgtagtt catactagaa ttattcccat ttaaagatgg 22200ggaaagtgat gtttagagag gctaagtgac ttgcccatgg ccaaagtatt agaactggga 22260tttgaacaca ggtagcctga ttcctaaata aatgaccact aacattaaaa agacataaac 22320atagaggtgt ttgttgcaat gttagtcata atacctaaaa attgtgaact agttaaatgt 22380ccaatagtag taaaatggtt cattaaccat ggtacaccca tcaatataat gttatattgt 22440catttaaaat tgtattccca agtttcgtat caggaaaata taataaattt ttgaaaaatg 22500taatgggata tattgtattt ataatacagt aatcactatg taaccaacat ttttatttcc 22560atcaaaaatt agttataaag aaatagaaat ctaagaagtt taatagatat taccattgaa 22620cactagattt gtattagtat cttttatatt tatctctctc atttcctata ttggccatgt 22680ttacttttaa aatagtaaaa gtcataagct ctattttaaa aataaatgtt agtttattaa 22740cccaagaata atagctaact ataaattcat atttgataaa ataaaaagat gacattcatc 22800ataagggata catacctgtg ctagcatatg gcatttttaa aatcacatga gtaatttgta 22860atcatcatag aaatattaga aaatacaaat aagcaaacac atactgaaaa tttagtgttc 22920ccaaatctaa aacagagaca ctgttttttt cttttgggat tgtattttag atatgttctt 22980aagttataat aaagtaaata cttaaaaggc aaattgatac attaaggatt tcaaagagta 23040aaactttttg ttaagctttg atgttcttta gaaagtttag attattccac aagtcactgt 23100cgttgaaaga aaagtagtta cagtggggtc ttatgggata aggcattacc atttgttcag 23160ttgagagaca gctatcacta tgttttaagc attgttcata tattagctca tttaatcctc 23220atagcaacct tatatgatag gtacctttat tagccccatt ctgcttagga agcaacagaa 23280agagtatgta ttttgcctaa ggttgcacaa taagttaagc tggggttcca attctagcaa 23340gttggttcta gagtgtatgt tattaaccat tatgccgtag tgcctgcaag tagatctcta 23400gatgtcagaa atactcatct tcctctggtt acctggttgt tataaatctt tatgcttaat 23460acttatgtca ttatatgtaa atttcgtatt aaacactcaa cttattcaga agaatatcag 23520gtaagtgtag gttaaggctg ttttctatca gaaatcatta tatgtatata tattcctcag 23580ttcttatgtt gtttagtttt tctaaaatgt caaattttat aatatatgga gaagtataaa 23640tgtatattag aaagattttg tttatttgtg taatttgtgg cataagaaat atttgcctca 23700agatttggtg cttgtttagg tagttgctgg cattactttt ggaaatgtca gtaaattttc 23760atactgtctt ggaatttttt caatttttac attttattag taaatgtaat tacaggttag 23820taaatcactt atttgaacct gtttcctttg aaagttttat atttttattt ggaaatagaa 23880aaaccttaat ttcctctcgt tgggcagtat ggtgtcaaaa gcttgggctt tggagcttcg 23940tatataatct gggttcaaat tataacttac tagttactaa cttgggcaaa ttacccagtc 24000tttttgactc tcaatttctt tgtctatgaa atgtaatact atttaggatt gttaggatta 24060aatgagaata tatttggcat actgtctggt acatgatact taacaggtac tagttgtcta 24120catctttcta acttaggatg gatgccgatg tcttgggtaa catctcaaac tttatcagta 24180aggaaggtga gaatctgaag aaaatgaaac cttaaaaaga ttgaattcct ggactccatt 24240taaaggagta aatagctcac gaacaagact tgctgctctg caaagtcttc catgttgatc 24300ctggtctttg actccttatc tgtctgatta aattgaattc gctgccgtgg catccttaaa 24360gctggacctt actttgtcag tcctgccttc tccatgttgc tttgtgtgta agcttcactg 24420gactgtttgc tttttgctga ttattttatg tatttccata tgtctacttt agcctttgct 24480tggaatgttc taactgctct tgtttcttcc tctgtttact ggtttctgac ttaactctta 24540aggattatct aatatattac ctacttggtg aaggtttatc tgtgtcccca cagaattaat 24600ccttccctct ttaactctta agctatctta ttttttatct aatcgggtct ttggcacaat 24660tataggtctg tgtgtctgtc ttccctaata gaataggaaa gccttgagga cagtagtctt 24720tgcttactta gccttatatt ctcagtgccc cttgtgcaaa acgttcaata tatgtttgaa 24780tgattatgtg aatgaaggag ggggccagct gaatttactt taatgattat gtaacaccca 24840tttatgtata gttatagact tgtctgaaat gagttaaatc ctttgcaacg tttgctgcta 24900tgctttgaat gtctcttaca aaactcatgt tgaaatttaa ttgccattgt aatggtatta 24960agagatggaa cctttaagag gtgattagat aatggtatct ctatccttat gaatggatta 25020atgctgtttg aatggtagtg gatgagttat cttgggagtt tggcctcctc cgtctcacat 25080gcttccttac cttctgccat gtggtaactc aacacaaaga tccttgccag atgctgatgc 25140catgctcttg gacttcccag tctccagaac cgtgagccaa atacatttct gttcattcta 25200aactacctgt tttgtggtat tctagtaaag caacataaaa tttactaaga aaactggtac 25260caagagtgtg attgttgcca taacaaatac ctgaaaatgt tcaaatggct tgggttctgg 25320ctagagaagg atggaagagt ttggatgaac aggctagaaa aagcctgtat tgctgagaat 25380agagcattaa ggacaattct gatgaggatt cagaagaaga gagctgtagg gaaaatctgg 25440aacttcttag agagttgtca tcagttggta gaactataag tggtaaaggt ctttctgatg 25500atatctcaga aatgaagaac aagatactgg acactggagt aaaggccatc cttgttaaat 25560agttgcaaag aacttggcga aattatgttc atatcctaag actttatgga atgcagaatt 25620taagagtgat gaactaggat atgctgcaga agaaataact cagcagcaga gcatttaggt 25680tactggatgg ctacttttaa ccacttaaac taagctgggg gaagggaatg acttgaagac 25740agaatttata attaaaagag aggcagaatg gaaatacttg gaaaatttgc agcctggcca 25800tagtaaagaa tgcaaaaggt atgtttagga gagcaaacca agggtgtggt ccaggaacca 25860tttgctgaag agattaatat tcctagagga gacccaaggg ctatttatca agacagtgga 25920aaaagacccc agaggcattt tggagatctt tgaggctgcc tgccccatca caggcccaga 25980gctctaggag ggcagaatgg tttgtggctc aggtggtcct tcacaagctt gctgcccagg 26040gctacctcag ctccccatat ttcaacccag tgggccttgg ctgtcctagg tctggttcag 26100aggggcccag gtgtggctta ggctactgct gagtactcaa atggtaaacc ttggcagcgt 26160ctatatggtg ctaattctgc aggctcacag gatgaaagag ctgtgggaga catggctacc 26220accaccagga tttcaaagga tgatggggat agtctgggag agacttgcca caggcttgga 26280gcctctgaag ggtggaaatg tgggttggag tcactacaga gagtccctac tagggcattg 26340cataatggag ccatggcagc aggcccacca ccaaagcttc agaactgtag agctacaagt 26400atacagtgcc agcctgggag aacttcaggc ttgagaccct aacctgtgaa agctgcatgg 26460gctaagtaca gcaaagccat ggaggtgggg cttcccaggg tctattggga tgaaacgaat 26520gtattttgta ggtgagaagg acatgagttt tgaggcccag gggcagaatg ctatggtttg 26580gttgtttcct tcaaaactca tgttgaaact tcattgccat gtggcattat tatgaggtgg 26640aacctttcag aagtggttac attataaggg ctttgccttc attaatggat taatgccatt 26700attgcaggtg tgggttagtt atctctggag tttggccccc tttttctcta tcatgtgctc 26760atgccctctt ttgccatgtg atgccttcta ccatgttatg atgcatccag aagactctca 26820ccacatgcag ccccttgatc ttagacttct cagcttctag aactgtgagt gaaataaact 26880tcttttcttt ataaattacc ccgtctgtgg tattgtatag cagcagaaaa tagacatcag 26940cctgaagttc ccccaggctg gcattaaata ctaattatta attagtattt atagtgcaag 27000gataaattca agtttagccc tggttagaat gaccacattt caagggaggg gctttgtact 27060tctgtgcata tctgtaagga taaaaatctt aatactattc tcactgaaat taatggttta 27120ggttaggtaa ggttgttagt gctaataatt atttctttta ataaaatatt cttagttgcg 27180ttgttcaaaa aacatagatg atttgaattt atattttttg gccaaaatat atttataatt 27240ttgagtagga attccagagt attggtagct ataaccactt tgggttccct gccattgctt 27300ctggtgcctc attttttctg acgtcttcca ttttcttaca tttgtcttct aaggtggagt 27360taagattact cagttaagat tatttcactt taggcctctg ctgtcttctg cttttttttt 27420taaaatgaat ggatataata tcccaataca ttttgataat tgaacaacag ctacattttt 27480aagtgaggct actttcttct aattttttaa atttattttt ctcagttttt aaaaaaaatg 27540tcagattggc taagagttgg ggcagctttc ttatgtgaga gtagtagatg acagcaaata 27600tttgtgatgt taaaatgata atcctaatag ttttctttta gaatctttat aataaaaacc 27660ctttgaggct gagggtgaat ttgtatgttc ctaaagtgac aaaaaatgtt cttggggcat 27720agtaatttaa atcttagatg cttttattag tataattttt tggtagaaat ttggcattaa 27780aaaatgcata cagagctttt tctacataca gggcaagaca gcattttgtc atggcaatta 27840gtaaatagat aattataaaa catctaattt taagcaattt gttacagaaa cgatacaggt 27900acattgtggc aaaaatagaa gatacaaaac aagcaaatat gagaaaaaat atacatgcca 27960ccacctatat atgactttta gtactttata atcttttgtc tatatatgtc tatatggata 28020tatacgtatt ctttttatcc aaatggtatt acattgtaca ttctgttttg aaacctgcct 28080tttttagtca tttacatcta cttttccatc tcagtaactt ttcatcttgt gtaatgcccg 28140gatcacatta aaatgtttcc aattagctca aaaatgtcct ttatggctgg tttggctaaa 28200acagtatcca ggccagcatt gcacctatga aattggctgt taggaatctt gtatctttaa 28260aaatcaaggg cagcaaccca tcctcccgct tcccccacct cccaccaccc ccccaccttt 28320tttttttctt aaagatactg gcttgttgaa gagagaatgg gtcatgtcct acaaactgtc 28380tgaatttgtc cagtttgctg tctcatagtg tcatttagct tgttttatcc tatgtatttc 28440ctgcaaatta aaatttgtat ctgaatcctt ggtggatttg agttgaagat ccttaaccat 28500catagatgat gctgtgtcct ttatattgca tgtcagaagt tacatgatct ttacttgatt 28560catgatgagg agatggccac tgaaattgga cagtgacagt cttttctgcc cattgttcaa 28620ttatattcgt ctctttacat tagaaagtat tttgggtagt agtattttgg tgctgtaaga 28680aagttcattt tctcatcaac cactcaccta ttggtttaac accatttgtt gatctttgag 28740tatatcagta atttcatcag ggtttgcaaa ataagacttt aaattctatt gttttacatt 28800attaattgat gttttttgat aaggtagaac ttgtgaaatg ggactatttg tttgtcttta 28860aatacagtct ctataggaaa gacaaaataa atacttaaat ctcactcttt accatttttc 28920aaagtgaaga actattccgt taaccacctc aaaagatgat aaataaaaag ggtattttta 28980gttgtttcaa cttttttttt ttttttgaga tggggtctta ctctgttgcc caggctggtg 29040tgctgtggtg caatcatggt acaccgaagc ctcagtctcc ctgggctcag atgattctcc 29100cacctcagcc tgggactaaa ggtgtacacc accatggcca gccaattttt ttgtattttt 29160tgtagagatg gggttttgcc atattgccca gcctagcctc aaactcctgg gctgaaggaa 29220tccacccatc tcagcctccc aaagtgctag gattacagac gtgaccaaca atatccggcc 29280ttaacttttt tcttttgagt gtctttatag actcaaggac ttttatttaa ttcagggtgt 29340tagtaccatt taaatgtttt ctttgatgct cagattatca cagctagtca tttggacctt 29400tataccacct cctatgtcca tttgatatag gccattaatc tctataagcc ttcctccttc 29460tcttggaatg aaaaggtatc ctaggctcac ctgtaccttc cctactccag acctggcatt 29520aagtcttttt ccaaggagtt tggtaccttt tagtttatta tgatattaga gatgaaaatc 29580tgtgttctag gaatgtttat tactgctaga gtgatgttgc ttttaggcca tttcagagaa 29640aagacctaga aaacagattt ttacaaacat gaattcatac tgatattttt agttttttac 29700atgatttctt gattttacaa tattatctgc tttcttaact taaaattatg aaccttaaag 29760tcattagcat aacttctttg cttatttcta caacataaag aaaatagtcc tggtgcggtg 29820gctcacactg taatcccagc actttgggag attgaggcag gtggattgct tgagctcagg 29880agttcaagac caacctgggc aacatgatga aaccttgtct ttacaaaaaa ttagctgggc 29940atggtggcat gtgcttgtat tcccagctac tcaggaggct gaggtgggag gatcacctga 30000gcccaggagg tcaaggctct agtgagccat gatcatgcca ccacactcca gcctgggtga 30060caaagtgaga ccctgtttca gggggaaaaa aaagataaaa

tagtttgagg aggctggatg 30120tagtggctca tgcatgtcat cctggcactt tgggaggtca aggtgggagg atggcttgag 30180cccaggagtt tgagaccagc ctgtgccaca tcatgagacc tggtctctat ttaaaaaaaa 30240aaaaaaaaga aaatagtttg aggatatcaa taatgatatt actagaatca gtaaaactac 30300caaaagaagt ttaaagtttc ttcctagtgt ttttttgttc ttagaatact tcctaccaag 30360aagtgcagta aaagtgcagt gtccaaatag cccttgtaac aaaacctttc tctttctcct 30420gggtgccaat ttgacattta atcagttttg tttctagcag tgttcaattt attagattat 30480aagtcttttt tttctttata ttattctaag atcaaaaata tataaagata tacacaggag 30540tcctgctgct acctgttctt gctatgcttt tccccttttc ttccctttct ctgtgaagca 30600gccattttta ttagtttctt gtttatcact catgcatgca tatgtttatt gaggatgttg 30660acattcaagc aaatatatgg gttaacattc tttttgtcat ccctatacga aagatatacc 30720cagtatactc tattgggtgg gtttttttcc ttaaaatatt cagtagatct ctccagttag 30780cacatagtta tcttatagat agaacatata catataacct tttcttaaac tatgctatta 30840aaataatagc tttcagtaac ttgataatta tttttggatt gaaaatacta ctgaaatcaa 30900ctcaatcatg tgaaagctgc agaaagaaaa agacctagaa aaagggcatt ggattaggtc 30960aactttgaat tttatttgga agataaatga gtccagaagt gagtgggcag agattattgg 31020agttggtctt gaaatgaggc gttaggcaga ttgactgggc tggtgtgaaa ggtctgtcag 31080aaaatcatga gattagattg aggtacctca aaaaatgaga gctggtatga tgagtgggta 31140agaatcataa aagcgtagag tgttgatgat ttttatagtt tataaatggt tcttgtgtgt 31200agagttttgt ttttatgcta gctatagtct gtaacataat tcactataat gggcatgcta 31260aatatccatg acagttgacc cttgaacaac acagagggta ggggcgccta cccctgtgca 31320gttgaaaatt cacatgtaac ttttgactcc ccaaaactta atatttagcc tatacttgac 31380tagaagtctt actgatgaca taatgttcgt taatacatat tttatatatg tgtcagatag 31440catatttgta taataaagta agctgcagga aaaatattaa aatcataaag aagagaaaat 31500atacttacta ttcattaagt ggaagtggat cctcataaag gtcttcatcc tcactgcctt 31560cactttgagt aggccgagga gtaggagaga gaggaaaggt cagacttgct gtctcatggg 31620tggcagaggt agaagaaggt ccacatacaa gtggtccgac acagctcaaa ccggttttgt 31680tcattggcca actgtagttt gattgaaagt aataataaat gaagtttctg cctcagttca 31740gtattatcaa gtcatagata gcaagggctg gaagaaacct tagtagtaat ctctttgagt 31800ctaattatca tgtagaatag gaaattgcgg tctagaaagg ttaagtgact tgtccaaatt 31860acacaactag ttagagacat agccagctct taaatctgac ttccagattt tcactgtgtc 31920ttcttttttc tgtaacgtgt tgcctttttt agccatgaaa aattagaagt tgaactcttg 31980tcttttcagg caggtgtcaa ttttggggtt ttgttttgat ttttggtttt tgacataaag 32040tactttagtt ctgtgatgta taaaccgtga gtttctgttt ttctcatata cctgaatact 32100gtccatgtgg aagttacctt ttatctttac cagtattaac acataaatgg ttatacataa 32160atacattgac caccttttat tactccagct atagtgggga aaactttctt ttcataacta 32220gctaatgttt taaaaagtat tcttttagtt tgattgctgc atatttcaga tatttctttc 32280cttaactaaa gtactcagat atttatccaa acattattgc tatgggattt cctgcagaaa 32340gacttgaagg cgtatacagg aacaatattg atgatgtagt aaggtaagaa tgctttgatt 32400ttctatttca aatattgatg tttatattca tgttgtgttt tcatttagaa aagatttcta 32460agccacagaa aaagatactt tgtgatgtaa actattattg tagtgctcta taatcatttt 32520ttggcttacc gtacctaatg gacttcaggg ggatacagtt catttgataa gaactgacct 32580tatacattac ataatcaggt acttatgtga tatcatttcc tggactccat aaaatgctgg 32640tcaccaggtt taatacctgg attccattac agtgtgattt ttgtcttatt tcatagttgg 32700ggattaggct taaaatccta gagtggattt attcagttaa atttattcac actaagatgt 32760agatgactaa tactgtatat ttttatgtag accaaatttt aaggtaccac tgtgcatatg 32820tataccaact acctgaagaa gtatttggtt ggtacaagag atatagaaag gaatcgctgg 32880gtgtaccaag gctaatcagt tttataattt tgcataattt tctaactgcg attatcattt 32940agtttagaac aatttatttc tcaaggccca tgtaaatatt atttttaaaa tatacagtct 33000taagaattca tggcatattt tatgaaagga ggaattcatg tctgatgtgc aaatagtctt 33060aacatatttt ctaatttcag agcaaaaata tatatgtatg aataaattaa ctgtaaattg 33120tcagtaggaa ccttaagaat tcgtggcgta ttttatgaaa ggaattcatg tctgatgtgc 33180aaatagtctt aacatatttg ctaatttcag agcaaaaata tatgtgtatt aataaatcaa 33240ctgtaaattg tcagtaagaa ccttaatggc tttaaaagtt aaaatttcag gtcaagcatt 33300gtggtgtgct cctgtagtcc cagctacttg ggaggctgag gtgggaggat cacttggctt 33360gaacccccag gtagagggta gaggccagtc tgggtaacac agcgagaacc catctcttaa 33420aaaaaaagtt taagttgtgg attatttcct ttacactctt tcattagtat ctttcctgga 33480gactttcaat ttaaatactt ggtgcttatg acaattagat gttaaaatgg atgggaaagt 33540actttgtaac ttataaagca ttatgcagat gtagactcct tttataatag ttgtgtaagt 33600atataagaca acctacattc ttcatgagct agccataagt tttagcaact tgctttgaac 33660cacggtagat ttacaatttt ctgtagtatt gagttgtgtt catttagaat tttgtaatat 33720ttatattgaa aatcaaattt ttgtacctac aaaaactaca aaaaatcccc ctagttttta 33780tagtttctat taaaattata gctggtacat agggatgcca gaaggactgt ttaagaagct 33840gaaaatagag aaatgaattt atcttctcat agttaggcag ggcacagtag aaggatgctt 33900aacattgcaa gctgatggga acagcaggtt gatatagctt gtgataacac ttctaaagaa 33960aaagcaatga gccatagaaa aaagaaaaag atacattttg aattaaggaa gatggtgaat 34020ctgggaagtg agcagtacag tcaccagacg tgtatcctct cctatggtac agaagtgttt 34080attgggtctc tttatggcct gcatgatata tcccacaaga tgacctactt cacattattt 34140taattctgta ttcaactaag cactaattca acccagccag attagtactc ataccaaaaa 34200agagtgaata ctctgaatag agggcaggtt ttctgattat ggtgagaata tctttgtggt 34260aaattaatct ggtgtgctag tttttacgtt ggtctcttct cagtgtcgtt agtcactgag 34320gctgattgat catcttttag gttactgata aagttcctgt acagctgatt ttcagacctt 34380agattgcaat aacttcacca agaaaatact tcattgggaa gcattttggt ccttccattt 34440gattcataac tcttaccttt atgcctctga aggaaaagat ttatacattc agcttgtaat 34500tagtaatcaa gactgaggtt tagtctatct agcttcacaa tctatctagt ttgttttgtc 34560tagccatatg atttcttcaa atatgccatt tcttaaaaaa aaatgtttta tgtatcccga 34620ttaatattta gccagtggtt cttttagccg atggatcttg tcacctctta tgatactatt 34680aatagcatgt caacatgaag aattatctgc tgaatataat agctatgctg tccttgtttc 34740cttttgtctc attctttttt gattggggga taattggcca ataaagcttt gatagcctct 34800attgcccagg cccctcctct tcttttatga gagaaaggat gaacagtgac cagaaataaa 34860ggtattgttt ttttctatca actaaaatgg aaataaataa ttcctaagta atttgcctgt 34920taggattaaa gtctccaaga gaatggctgt gcctagtacc taagtgatta atttccttga 34980ttggttcaca ttatattgag gatattagta atcagtagtg attccttttt tggttcaaag 35040atgatagtgt cacagtgaaa aatgttttta aaatttttgt atacttaatt tttctgttaa 35100cgaaagtatt ttcagttgga tttttgtttg ccctctctat tagaatgccc aaagaatatt 35160taaaattttc cttttctctt atactgcata tttttcctgt gatttttccc caaacggaaa 35220atactctgca gagattagac tttgttattg ttgtactaca tcattgcttt gactaaaata 35280aactcagatt gcaaatacct tcaagcttac attgctcagt attttttttt tttttttttt 35340ttgagacgga gtctcactct tgtcgcccag gctggagtgc agtggtgcca tctcagctca 35400ctgcaacctc tgcctcctgg gttcaagcga ttctcctgcc tcagcctgcg gagcagctgg 35460gattatagat gcccgccccc acgcccagct gatttttgta tttgtagtag agatggggtt 35520ttaccttgtt ggccagtctg gtctcaaact cctgacctcg ggtgatccat ctgtctcggc 35580ctctggaatt acaggtgtga gccgccacgc ctggctaaat tgatcagtat tatttaactt 35640tgagggatat gatttgttat ggaatgcgaa gttttatact tgaggtactc agagtccttt 35700tgagacaaat atttaacttc tccttttgag gttaccgcct acgattggga attaatgtaa 35760aaaataagcc aaaagaaagt gagggaaaag tgaaccaagc tgtaattttt ttactctttt 35820ttattgttgt tgttattgtt gctgtttttt actatcttga ttgcaacagt ttggcttata 35880tatatagcat ttggaattga cagtaagaaa gccacatctc atagaagcta actattccca 35940aattgttttt ttcttctttt cctcttacta ctgctgtttt cctcctttct tgctgctaag 36000ctcttgtcct gacatgctgg taatatgaaa cagtgtttta ttcagataat tgattattct 36060gtaatatgta tgttaatctt ttttattaca ctttaagtaa tagggtacat atgcacaact 36120tacagattcg ttacatatgt atacatgtgc cgtgttggtt tgctgcaccc attaactcgt 36180catttacatt aggtatttct cctaatgtta tccctctccc aaccccccac cccaggacag 36240gccccggtgt gtgatgttcc ccgccctgtg tccaagtgtt ctcgttgttc agttgccacc 36300tgtgagtgag aacatgcggt gtttggtttt ctgtccttgc gatagtttgc tcagaatgat 36360ggtttccagc ttcatctatg tccctacaaa ggacgtgaag ctcatccttt tttatggctg 36420catactactc cgtggtgtat atgtgccaca ttttcttaat ccagtcagtc attgatggac 36480atttgggttg gttctaattc tttgctattg tgaatagtgc tgcagtaaac atacgtatgc 36540atgtgtcttt atagtagcat gatttataat cctttggata tatacccagt aatggaattg 36600ctgggtcaaa tggtatttct agttctagat ccctgaggaa ttgccacact gtcttccaca 36660atggttgaac tagtttacag tcccaccaac agtgtaaaaa tgttcctgtt cctccacatc 36720ctctccagca cctgttgttt cctgactttt taatgatcgc cattctaact ggtgtgagat 36780ggtatctcat tgtggttttg atttgcattt ctctgatggc cagtgatgat gagcattttt 36840tcatgtgtct gttggctgca taaatgtcta taaatgtctt cttttggaaa gtgtctgttc 36900atatcctttg cccacttttt gatggggttg tttgattttt ttcctgtaaa tttgtttaag 36960ttctttgtag attctggata ttagccattt gtcagatggg tagattgcag aaattttctt 37020ccattctata ggttgcctgt ccactctgat ggtagtttct tttgctgtgc agaagctctt 37080tagtttaatt agatcccatt tgactatttt ggcttttgtt gccattgctt ttggtgtttt 37140agtcatgaag tccttgccca tgcctatgtc ctgaatggta ttgcctaggt ttgcttctag 37200ggtttttatg gttttaggtc tacatttaag tctttaacat ttaagtcttt aatccatctt 37260gaattaattt ttgtataagg tgtaaggaaa tgatccaatt tcagctttct acatatgact 37320agccagtttt cccagcacca tttattaact agggaaccct ttccccattt cctgtttttg 37380tcaggtttgt caaagatcag atggttgtag atgtgtcatg ttatttctga gggctctgtt 37440ctgttccatt ggtctatatc tctgttttgg taccagtacc atgctgtttt ggttactgta 37500gccttgtagt atagtttgaa gtcaggtagt gtgatgcctc cagctttttt ctttctgctt 37560aggattgtct tggcagtgcg ggctcttttt tggctccata tgaactttaa agtagttttt 37620tccaattctg tgaagaaatt tattggtagc ttgatgggga tggcattgtt tctataaatt 37680accttgggca gtgtggccat tttcacgata ttgattcttc ctacccatga gcatggaatg 37740ttcttccatt tgtttgtgtc atcttttatt tcgttgagca gtggtttgta gttcttgaac 37800aggtccttca catcccttgt aagttggatt cctaggtatt ttattctctt tgtagcagtt 37860gtgagtggga gttcactcat gatttggctc tctgtctgtc tgttattggt gtataagaat 37920gcttgtgatt tttgcacatt gattttgtat cctgagactt tgctgaagtt gcttatcagc 37980tgaaggagat tttgggctga gacagtgggg ctttctaaat atacaatcat gtcatctgca 38040aacagggaca atttgacttc ctcttttcct aattgaatac cctttatttc tttctcttgc 38100ctgattgccc tggccagaac ttccaacact gtgttgaata ggagtggtga gagagggcgt 38160ccctgtcttg tgccagtttt caaagggaat gcttccagtt tttgcccatt cagtatgata 38220ctggctgtgg gtttgtcata aatagctctt attattttga gatacgttcc atcaatacct 38280aatttattga gagtttttag catgaagggc tgttgaattt tgtcaaaggc cttttctgca 38340tctattgaga taatcatgtg gttttttgtc tttggttctc tttatgtgat ggattatgtt 38400tattgatttg cgtatgttga accagccttg catcacaggg atgaagccaa cttgatcttg 38460gtggataagc tttttgatgt gctgctggat tcggtttgcc aatattttat tgaggatttt 38520tgcattgatg ttcatcaggg gtgttggtct aaaattctct ttttttgttg tgtctctgcc 38580aggctttggt atcgggatgg tgctggcctc ctaaaatgag ttagggagga ttccctcttt 38640ttctatgaat tggaatagtt tcagaaggaa tggtaccagc tcgtcttttt acctctggta 38700gaattcggct gtgaatctgt ctggtcctgg acttttttcg gttggtaggc tattaattat 38760tgcctcaatt tcagagcctg ttactggtct attcagggat tcaacttctt cctggtttag 38820tcttgggagg gtgtatatgt ccaggaattt atccatttct tctagatttt ctagtttatt 38880tgcatagagg tgtttatagt attctctgat ggtagtttgt atttctgtgg gatcagtggt 38940gatatcccct ttatcatttt ttattgcatc tatttgattc ttctctcttt tcttccttat 39000tagtcttgct agcagtctat caattttgtt ttttaaaaaa accagctcct ggattcattg 39060attttttttt tgaagggttt tttgtgtcct atctccttca attctgctct gatcttagtt 39120atttcttgcc ttctgctagc ttttgaattt gtttgctctt gcatctctag ttgttttaat 39180tgtgatatta gggtgttgat tttagatctt tcctgctttc tcttgtgggc atttagtgct 39240ataaatttcc ctgtatacac tgctttaaat gtgtcccaga gattctggta cgttgtgtct 39300ttgttctcat tggtttcaaa gaacatcttt atttctgcct tcattttgtt atttacccag 39360tagtcattca ggagcaggtt gatcagtttc catgtagttg tgcagttttg agtgagtttc 39420ttaatcctga gttctaattt gattttactg tggtctgaga gacagtttgt tgtgattttt 39480attcttttac atttgctgag gagtgagtgc tttacttcca actatgtggt caattttgga 39540ataagtgtga tgtggtgctg ataagaatgt atattctgtt gatttgggat ggagagttct 39600gtagatgtct attaggtctg cttggtgcag agctgagttc aaatcctgga tatccttgtt 39660aaccttctgt ctcgttgatc tgtctcatat tgacagtggg gtgttaaaat ctcccgatat 39720taactgtgtg ggagtctaag tctctttgta ggtcactcag gacttgcttt atgaatctag 39780gtgctcctgt attgggtgta tatatattta ggatagttag ctcttcttgt tgaattgatc 39840cctttaccat tatgtaatgc ccttctttgt ctcttttgat ctttgttggt ttacagtttg 39900ttttattaga gactaggatt gcaacccctg ctttttcttg ctttccattt gcttggtaga 39960tcttcctcca tccctttatt ttgagcctgt gtgtgtgtct gcatgtgaga tacatctcct 40020gaatacagca cactgatggg tcttgactct ttatccaatt tgccagtctt tgtcttttaa 40080ttggggcatt taccccattt acatttaagg ttaatattgt tatgtgtgaa tttgatcctg 40140tcattgtgat gttagctggt tattttgccc attagttgat gtagtttctt cctagcatca 40200atggtcttta caatttggca tgtttttgca gtggctgata ccagttgttc ctttccatgt 40260ttagtgcttc cttcaggagc tcttgtaagg caggcctggt agtgacaaaa tctctcagca 40320tttgcttgtc tgtaaaggtt tttatttctc cttcccttat gaagcttagt ttggctggat 40380atgaaattct gggttgaaaa ttcttttctt taagaatgta gactattggc ccccactctc 40440ttctggcttg tagagtttca gcggagagat ctgctgttag tctgatgggc ttccctttgt 40500gggtaacccg acctttctct ctggctgccc tttacatttt ttcctgcatt tccaccttgg 40560tgaatctaac aattatgtgt cttggggttg ctcttctcta ggagtatctt tgtggtggtc 40620tctgtattcc ctgaatttga atgttggcgt gccttgctat gttggggaag ttctcctgga 40680taatatcctg aagagtgttt tccagcttgg ttccattctc cccgtcactg tcaagtacac 40740caatcaaacg tagatttggt cttttcacat agtcccatat ttcttggagg ctttgttcat 40800ttctttttac tgttttttct ctaaacttct cttcttgctt catttcattc atttgatctg 40860caattactga taccctttct tccacttgat cgaatcggct gctgaagctt gtgcatgcgt 40920catgtagttc tcgagccatg attttcagct ccatcaggtc atttatggtc ttctgtacac 40980tgtttattct agttagccat ttgtctaatc ttttttcaag atttttagct tccttgcgat 41040gggtttgaac atcctccttt agctcggaga agtttgttac tactgacctt ctgaagccta 41100cttctgtcaa ctcgtcaaag tcattcttca tccagctttg ttccattgct ggtgaggagc 41160tgtgatcctt tggaggagaa gaggcactct gggttttaga attttcagct tttctgctct 41220gttttctccc catctttgtg gttttatcta cctttggtct ttgattatgg tgacctacag 41280atggggtttt ggtgtggatg tcctttttgt taatgttgat gctattcctt tctgtttgtt 41340agttttcctt ctaacagtca ggtccctcag ctgtaggtct gttggagttt gctggaggtc 41400cactccagac actgtctgga tatcaccagt ggaggctgca gaacagcaaa tattgcagaa 41460cagcaaatat tgctgccgga gccttcctct ggaagcttcg tcttgggggc acccggctgt 41520atgaggtgtc agtcggcccc tactgggagg tgtctcccag ttaggctaca agggggtcag 41580ggacccactt gaggaggcag tctgtccgtt ctcagagctc aaacactgtg ctggtagaac 41640tactgctctc ttcagagctg tcagagaggg atgtttaagt ctgaggaagt ttctgctgcc 41700ttttgttcag ctatgccctg cctccagagg tggagtctac agaggcaggc aggcctcctt 41760gagctgtggt gggctccacc cagttggagc ttcccaacca ctttacctac tcaagcctca 41820gcaatgatgg acgcccctcc cccagccagg ctgctgcctt gaagttcaat ttggaactgc 41880tacgctagca gtgagcaagg ctctgtgggc gtaggacctg ctgagccagg cacgggatat 41940aatctcctgt tgtgccattt gctaagaccg ttggaaaagc gcagtatttg ggtggcagtg 42000tcccaatttt cccggtatag tgtgtcacag cttcccttgg cttggaaagg gacatccccc 42060gaccccttgt gcttcctggg tgaggcaatg ccccgccctg cttcagctca ccctccgtgg 42120gctgcaccca ctttccaacc agtcccagtg agaagaacca ggtacctcag ttggaaatgc 42180agaaatcacc tgtcttccgc gtggatcatg ctgggagctg cagacaggag ctgttcctat 42240ttgaccatct tggaatgcca cctttttttt tttttttttt tttttaaggc agtttcttgc 42300tctgtcaccc aggctggggt gcagaggcat gatcacggct cactgcaacc tctgccttct 42360gggctcaagt gatcctccca cctcagcctc ccaagttgct gggaccacag ccacgcatca 42420ccaggcctgg ctaatttttg tgttttttgt agagataggg tttcgctgtg tttcccaggc 42480tggtctcaaa ctcctgcgct caagcgatcc gcctgcctca gcctcccaaa gtgctgggat 42540tacaggcatg agccactgca cccggccaat atgtatgtta atctcatccc tcaagctgat 42600actgaagttt ttcaatttat gttatttggt gtaaatctag gcagtcttta acaaaattgg 42660tgcttcatgt gtttaagagg cataacttaa gaattgtttg tttcttataa atcaggagaa 42720tggaggttta atagaggtga actgtctttc tcactgcaga acctttaata tgccactatg 42780cattgtaaat ctcccaagag tgagattcta gtatgatgct tttcttttcc ttttctgttc 42840tttccctttc cctctacctc ctttttcttt tctttgttgg tggcatgagt cctatattat 42900aaggaaatgc ttttagagta cagtcttctg atatatagtg atttttgaaa aagatttatt 42960tattgtcttg ttcactgtga gctttttccc ccatgtataa gcagctgtgt aatagattca 43020agagcacccc ctcgcccctt tttttttgag acagagtctc gctcggtcac tcaggctgga 43080gtacggtggt gctgtgatca tggttcacct cgacttctgg gctccagcga tcctcccacc 43140tcatcctctc aagtagctgg gaccacaggc gtgtgtcacc gtacatggct aatttttcta 43200tttttagtag aggcagagtt tcgccatgtt ttccaggctg gtctcgaact cctgaactca 43260agcagtccac ctgtctcagc ctcccaaagt gctgggatta caggcgtgag cctccactcc 43320cagtctcaaa tattcttttg aaatatttga aatatgttga tctctcagtc tttcaacctt 43380agttgtatgt tgattttcaa taaaaaggaa gtatttgttg ccctaacatc agtattggct 43440attcagttta aaaaaggagt taaagagatg ttatttatag gcaggcttca aaagaggaaa 43500gaatgatcag tttcattctc tgtttctagc atattctgac tccttctctc atattacctc 43560gtttttccca cattttttct ttaataaagt gaaattcaca taacagctaa ccattttaac 43620cacggaaagt gtacattccg tggcatttat taccttcaca gtgttacctc tacctttatc 43680aagtttcaaa acattttatc accccaaaag aaagccctgt tcttattggg tgcctcttgc 43740tttttttttt tttttttttt ttaaatcttg agacggggtc ttggtttgtt tcccaggttg 43800tagtgcaatg gggcgatctc atctcattgc aacctctgcc tcccgagttc aagcaattct 43860cctgcctcag cctcccgtag ctgggactac aggcacgcgc cacatgcctg gctaattttt 43920gtatttttag tagaaacgtg gtttcaccat gttggccagg ctagccttga actcctgacc 43980ttaggtaatc tgcctgcctt ggcctcccaa agtgctggga ttataggcgt gagccaccgt 44040tctggccacc tcctacttct ttcattagtc ttaattcctt agtggatttg acagtgttta 44100tattatctac accaatgcat ttttttgtat gttaataata ggagatattt attgggcatt 44160tatttacata cttatttgca tgtgtaactg ttgtatagcc agtttatgta tataatctta 44220ctaaatctct acagtaaatc tatccccatt tcatagatgg tttaaaagaa gttaacttcc 44280ccaagcatta cttttagtaa gtagtgagac tgaaggttga gttctggtct gtcagattcc 44340gaagttgttt ccttaggaac catattatct tgtgtacaac tcttgggcta atcttgttaa 44400tattctttat ttgacctcac actgttgatt cataccatgt ttaaaattga aatacattac 44460ctatatttaa aaattgagac ctcacataaa aagctatatt tctagctgct tttgaaattt 44520cgaaggatct tccaacactt ggctgacatt cctgcgtgac aaaaatcagc tggagctgtg 44580taatgtccga cctgtctctg tcaatgaaca gattattcat tgtcatttct catctgtttt 44640tgagattgta actttgattc tgtataactc atagaattag tagttagacc tatatatgtt 44700agttattaca ttatttgtat atgtacagac tgctttagac aatattgtag tgttatatgt 44760taattttatc aattaaaatg tgctatagga tcattgtaga gatcttgtct ctaattaaca 44820ggattcataa agagaaaaca aaggagagaa acatccagat tgaaaagacc tacatagtgc 44880caagcacaat aaaagaaacc ctatactagg caaattatta tgaaatttgt tttaggacac 44940cagaaataaa gataatatct aaaaactttt tagaatcgcc taggagggat taggaatatg 45000aatagcatct aacttgttgg cagtagaatt gaaggtagaa ggtggtaaaa catcaccttg 45060aaagttctgc attcagccta gaattctgta tccagttaaa ccatcaatca agtgtgaaag 45120tagggggaaa aactcaagga ctaaaaaaat gtgctcatga

agaacatttt ttttggacat 45180tacccagaag atgtggttca acaaaacaag ggaataaacc aagaaaggaa ataaaagagc 45240ttcagtaaac agagaatccc actcagaagc aacaaagaag aaagtcccag gatgacagct 45300gtgacacaag ctgaaaagtt accagttttg cttggagcag gagattagaa cttccgggaa 45360aataatcaaa ttgatagatg gatgttgaaa tatttggaga aaaatgtaat ggattcttgc 45420aaaactgagc aaattagaaa aaggaaacaa ttattagcat tggtggtttg agttaaccca 45480aaattgtgat gttgctattt taggggaatt aagataagtg aaacacgtat ggaatactgc 45540tagttttgta agtctccttt accatggcag gacatctgta gttaataaat ctgtaagaag 45600cagtattaag aataatattt taaaatacct aattaaatag gaggaaaaag aatccgagta 45660gttgagggtg attgcatctg gggagaagca ggaataaagg tttgaagata aatagggcca 45720gagatgagta attttgttac tgtgataata tattttatat atatgtgtat gtgtgtgcgt 45780gtgtatatat atatatgaga tatatctcat atatatgaga tatatatgat atatatatga 45840tatatatgat atatatatat aaaacataca gttcagtagc attaacatct agcattcagt 45900acatttacat tgttgtgcaa ttgtcaccac tgtccatctc tagaactttt tcattatccc 45960acactgacat accacaccca ttaaataata actcctcatt gctcctcctg ttagtaccaa 46020ccattgttct actttcatct ctatgtattt gactattcta ggtacctcgt ataagcggaa 46080tcacgtgata tctttttgta actggcttat ttcactaact atcttcgagg ttcatccatg 46140ttgtagcagt tgttagcatt tccttccttt taaaaggccg aataatattc cattgttatg 46200tatataccat attttgttta tccatttatt catcaataga cacttttggc tgttgtgaat 46260aatgctgcta tgaatatggg tatgtaatac ctgtttgagt atctgctttc atttcttttg 46320gatatgtacc caaaagtgag attgctggat tgaatggtaa ttctatattt aaatttttga 46380gaaaccacca tactgtttga tatactggct gcccaatttt acattaccac cagcaataca 46440ctagggttcc gaatttgcca catcctcacc atcgtgttgt tttgtttatg ttttttttta 46500ataatagcca tcctaatggg tgtgaagtct cattgtggtt ttaatttgca ttttcctaat 46560gatcagcgat attgaacatt tgcacatgct tatttggtca tttgtatatc agctttggag 46620caatgatgtc tcttgaagcc ttttgcccat ttatgaattg agtagtttgg gatttttaaa 46680ttgtgtttta gaagttcttt gtatactctg gctgggcacg gtggctcgtg cctttgggag 46740gccgaggcag gtggatcacg aggttaggag ttcaagacca gactggctgg tatagtgaaa 46800ccccatctct actaaaaata caaaaattag ctggtgtggt cgggcgtgat ggtgcacacc 46860tgtagtccca gctgttgggg aggctgaggc aggagacttg cttgaacccg gaaggtgggg 46920gggttgcagt gagctgagat tgtgccactg cactcagcct gggtgacaga gcgagactct 46980gtctcaaaaa aaaagaaaaa gaagttcttt gtattctctg aatattaatc ccttattgga 47040tatgttattt gcaaatattt tctcccataa agaatgggtt actttttcac tctgttgatt 47100gtttcctttg ctgtgcagga gctatttagc ttgaaaaaat ccaacttgtc tgttttcttt 47160tgttgcctgt acctttggtg tcacattcaa gaaataattg ccaaattcat accatgaaac 47220tttcccccat gttttctcct gaaggttttt atagttttag ctctcacatt taggtgtttg 47280atccattttg agttaaattt tgtatataat gttatgtaag agtccagctt cacacttttg 47340gatgtgaata tctagttttc ccagcattat ttgttgaaaa gagtgtcttt ttccccattg 47400aatagtcttg gcactcttgt tgaaaattat ttgacaatag atgcaagggt ttatttttgg 47460gctctctcga ctattctgtt agactatatg tttgtttttt tatgtcagga ccaccctaat 47520tttagtactg tagctttata gtaaaatttg aaaccaggaa gtatatgtct tgtgtattta 47580tttacttatt ttttgaaata gcatctggct ctgttgccca ggctggagtg cagtggcaca 47640atcttagctc actgcaacct ccacatctga ggttcaagca atcctcccac ctcagcctcc 47700tgagtagctg ggattgtaga cacataccac catgctcagc tagtttttgt atttcttgta 47760gagacagggt tttgccatat tgcccaggtg ggtctcgaac tcctgagccc aagcagtctg 47820ccctcctcag tgtcccaaag tgttgcgatt acaggtatga gccaccgtgc atggccccaa 47880cttcttatat ttcaagatgg ttttggccct tcagggccct ttgtgagttt taggatggat 47940ttttttttta acttttaagt ttaggggtgc atgtgcaggt ttattacata ggtaaatttg 48000tgtcaaggtg ttctgttgta tagattattt catcacccag gtattaagcc tagtacccat 48060tagttatttt tcctgagctc gcctcctccc acctggattt ttttttttat ttctaccaga 48120aacattgttg ggattttggt agggattgta ttagtctgta gattgcattg aatagtactg 48180acatcttaac aatattaagt ctttaaagcc atgaacacca gatgtctttc catttattta 48240cgtattcttt cttttctttc agcaatgttt tgtaattttc agtgtacaag tattttacct 48300ccttggttaa gttaattcct aagtatttta ttcattctga tgatcttata aatctgtttt 48360cttaatttcc tttcctaatt gttcattctt agggtataga aacacaactg attcttcgca 48420cattaaattt gtgccctgct tcttcgcggg tttgtttatt ctttttttgt gtttgaaatc 48480cttgaggttt tctgcatata agattatatc atctgcaaat gagataattt tacttgttcc 48540tttccaattt gagatgattt ttattcattt tcttaatgct ctctcataca ttcaatacta 48600tgttgaatgg aagtggtgaa agcaggcatc ctgtcttgtt tctgacctta taggaaaagc 48660tttcaattct ttgccattga ctatcatgtt agctatggga tttttttttt ccccccagat 48720agagtctcgc tgtgtcgccc aggctggagt gcagtggtgc gatctcggtt cactgcaccc 48780tcctcctccc gccaggttca agtgattctc ctgcttcagc ctcccaagta gctgggatta 48840caggtgtcca ccactatgcc cagctaattt tcgtattttt agtagaaaca tggtttcacc 48900atgttggcca agctggtctc gaactcttgg cctcaagtga ttcacctgcc tcggcctccc 48960atagtgctgg gattatagtc agccaccatg cctggccact gtgggatttt tatatatggc 49020cttcattatg ttgtggtaat ttctttttat tcttagttta ttgagtgttt ttatcataaa 49080atcttgttga attttttcaa atattttttc tgtgctagtt gagatgacca tgtgatttgt 49140tttcttcttt ctattaacat gatatattgt ttttcatata ttgagccatt tttgcatccc 49200aggaataaat tttacttggt cttcgtgtat aatccattta ataagctgtg gaattcagtt 49260tgttggttct gtgttgagga ctttatatca atgttcctaa gggctactgg tctatagttt 49320tcttttgtag tttctttgac tttgctatca gggcaatgct ggcctcattg aatgtgttag 49380gaagtgtttc ctcatccatt tttggcaaaa ctttgggaaa aaacgatgtt ctttaaatgt 49440ttgatagaat tcacagataa aaaaatcaca tctagggctt ttgtctggaa tttttttatt 49500gttattattg attcagtctt gttactagtt ataggtctat tcagattttc tttttgtgtg 49560tgtgattcag tattagtaca ttttgtactt ctcgttattt ctccatttaa tctatattat 49620ctaatttgtt ggcatacaat tgttcatagt actgtcttct ctttttttta aacttctgtg 49680cagttgatac taatgtccct acttttattt cagattttag taatttgaat cttctttatc 49740ttaatacagg taaagctggg tcaattgtta aaattttttc aaagaaccag tttttggttt 49800cattgatttt tctctgttat ttttctatta tttatatcct ctctaagctt tgttatttcc 49860ttcatcctgc tagctttggg tttattagtt tgttcttttt ctagttcctt aagatttgaa 49920gttggattat tgatttgaga tcgttttcaa ttaaatgtgt acaactacaa atttccctct 49980taggactgct tttgctgttc tgtaattttt ggcatgttgt gtttttttgt tttaatttat 50040ttctaagtat tttctaagtt cccttgtgat ttcttctgcg tttaaccgtg tattttttaa 50100tttccacagt tggtgaattt tctacttttt cttcagttat tgattttatt gattttcagt 50160ggcatcctgt tgtgatagaa gatactttat ttggttccct gttttttttt taaaacagag 50220tgttgctctg tcacccaggc tggagtgcag tggtgcgatc ttggctcact gcaacatcca 50280cctcctgggt tcgagcaatt ctcctggtct cagcctcccc agtaggtagg attacaggca 50340catgccacca tgcccagcta atttttgtat ttttagtaga gacagggttt cgccatgttg 50400gccaggatga tctcgaactc ctgacttcaa gtgatccacc cgccttggcc tcccgaagtg 50460ctaggattat aggcgttagc caccttgtct ggcccagatc ctctattttc ttgcctatat 50520tctgactggg tgtttttgtc cattattgag agtagggtat cgaagtgtcc agctgttatt 50580tcagaactgt ctgtttacct tcaattctgt caattttttg cttcatataa ttgggtggtc 50640tcttattagg catgtaaatg tttttgatta ttatatcttc ttgctatatt gacacttatt 50700gatgtgtaat atcttttttg tctcaacctt gttttgattt agtttgtcta atattaatgt 50760agctacctgc actctcattt ggttattact tgtatagaat gtctttttac atcccttatt 50820tgtgtctttg gatctgaaat gagtctcttg tagacagcat atacaatctc ttgtagattg 50880tgtttttctt atacattctg tcaaactctg ccttttgatt gaagagtttt atcatttaca 50940attaaagtaa ttattgataa ggatttaact gctgccattt tgctgtttat tttctatatg 51000ccttacagct tttttgtccc tcatttcctg ccttactggc acttgtgttt agttgatatt 51060ttgtggtgaa gtgttttaat ttccttgtta tttccttttg tttatattct gctattttct 51120gtgtgtgtgt gtgtgtggtt gccattgggg gtcacattta atgccctaaa gttataacac 51180tgcaatttca atttatacca atttactttc aatagcatac aaaaattcag ctcataagat 51240tcagtccctg ctcccttcca gttattgatg tcataaaatt acatttttat gtattgtgtg 51300tctaaaagcg tagactaata attgtttttt aatgcagtag tgtcttaatt tgtggaaaac 51360aaaaagtgga gttgtaaacc aatgttacaa taatgctagc tttggtaatt gctcatgtat 51420ttatcatttc tcagatcttt atttcttaat acagcttcaa gttactgtct agtctccttt 51480tatttcagcc tgaaagactc acttcagcgt ttcttgcagg acaggtctgg tgataatgaa 51540ctctctcaga ttttgttaat ctggaaatgt cttaatgtct tcatttttaa ggacattttt 51600gctggatata gtattctcag ttgacaggta tttgtgttta tttgtttgtt tcctttcagg 51660attttaaata tatcatccca ctgccttctg tccttcaaga gttctgatga gaaatctgct 51720gatattgagg atccctttgt atgttacaag ttgcttctct cattccatgt tcaggattct 51780cttttttcat agtttgatta taatctatct cagtttttcc tacttggatc tctgagtttt 51840tcctacttgg agttaattga gcttcttgaa tatttatatt catgtctcat caaatttggg 51900aagtttttga ttaatatttc ttcacataat cttttttgcc ccttttctct ctttttattc 51960tgggattccc agagtgtgta tgtgttggtc cacttgatga tggtgttcca caggtgtctt 52020aggctcttgt cttcaatttt ccttcagttt tttttttctg ttcctcagac acgtggtatt 52080ttcagctgtt ctgccttcca agtttactga ttcttctgcc tgcccaaatt ggcttttgaa 52140ttcctctagt aaatttttat ttcagttttt gtacttttca gctccagcat ttattttttg 52200atttttttat gttttctctt tattgatatt tcaattttgt tttttgacat tatccatatc 52260ttcctttagc tttttgagca cctttcaaca tttgttttaa agtctatgtc tagtaagtgt 52320ctgccatctg atcttctcag gcacagtttc tgttaattta tttttttcct tttggcctat 52380acttaatgtt ttgcttgggg tagttttttt ttttggtatg ctttgtgatt tttgttgttg 52440ttgtcaaaaa ctggaatttg aatcttaaag agtggtaact ttggaaatta gatttttctc 52500ttttctctaa gatgtgctat tttgtttggt ttttttattt gttgtagagt atttctatgc 52560tgggtgtaat cttaagatct tctcggcctg tgtttttccc tgggcatgtg tagtgacttt 52620ctaaattgcc ctatgtatgc agttcttttg cagtagtatc ctccttaaat gtttggctcc 52680taaaaggcaa aataaataaa taaataaata aaaattaaaa attaaaaata aatcaaaggg 52740gtgaaatagc tctggatctt taaatccctg gagcaatttt ttcagccaat ggcagttaaa 52800taatgatagt ctgcctctgt gtcacatttt gatcagaagc agcaattagc aatcagaaca 52860cagattcctg atatttggag ggcaaggtct ttgttgccaa ccttgactct tacaaactgt 52920gtgcagggtg ctctgggaac atgtgcatgg ttgcctgctt tgagagtggg tgatgggtag 52980ccgcgacggc acaaagagct gaaattgact caaactaact gatttaccat tcaagtcttt 53040ccttagaaac tgaaaacctg aatagactcc agagttccag aatcacagat tctgcttaca 53100gtcgtctagg tggggagatg ggttcctggt acttctgatt ctgccatctt tctttgacta 53160attttttttt tttgttcttg agatggagtc ttactctgtt gcccgcccag gctggagtac 53220agtagcatga cctcggctca ctgcaacctc tgcctcccgg gttcaggcaa ttctcctgcc 53280tcagcttccc aagtagctgg aattacaggc gtgcaccacc atgcctggct aattttttgt 53340atttttagta gagacagggt ttcaccatgt tggccaggct agtctcgaac tcctgacctc 53400aggtgatcaa cccgcctcag cctcccaaag tgctaggatt acaggtgtga gccactgcac 53460ccagctctta gacaaatttt ttattccaaa ctttttttat tttatcattt gaaaggtata 53520tgtttattat tttgtcaaaa ataattttaa aacgtattct tgaagcttat ttagatctgt 53580ttcataggaa ctgtgaagaa agtaaagaat ttaaaaaatg aagacagatt ttctcaccct 53640gcttatgggt gcttctcgtg ctagcctttt gcaagtgtcg ggaagtgtaa cctgcaggag 53700gcatcagggc tttgggcctg catggtctga gtgctgccct gtgagtttca gaaggcgcag 53760caacctgtat acctgaaagc catctctgct ggggcaggta cctagtgtcc ccacctacct 53820gggtcgtagt caggccctgg gcaagcctgc tatgcttttc cttccctaat ccctcagggg 53880tgggatagag agcacagtgg cctcccaggg aggtagaagc tgctccagac taacaatcag 53940agctgccagt tcttaatccc caagaccgcc agacttcaca aagacatacc gaggtctgtg 54000ctgtcagtgc cccactacta cactccctta agtagcccca cattcttgtg cttgtttctt 54060ttttctgctc tctttccttg cccaggtaag aggtctgccc ataagggata ttttgcagca 54120tgtgaagctt tttaaaaagt taggcttatt gaagtataat ttacacacaa agtacaaaaa 54180aaaaaagact gtgttctcaa atctgtgagt cattaatggg tttagatgtt tatatattga 54240aattattgga agtaaggtat gtttatatta gaaagatttg tagtctagat tatccaagtt 54300ttgggagtat tacctctctg cttttgttta tctacttttt tagtctctac tttccaagta 54360tctataggca aattttccca tttccctttg gaaagtgctg ttttcttgct ttttttccgc 54420ctttccattg tgtcagactt ataaggcaat cagccaactg tgggcatgaa atccttggga 54480ggaaagagaa ggaagtggga ggggcagcca tggtgaatgt ttccctaagt tatagtcaag 54540ttctttgaga gaacataacc tcatcccctt tttaaactgt tgtaatactt tcttttaaat 54600agattgttta ttctcctgca agtctcacag ttgttcacag tggtaggtaa gaaatcataa 54660agttcaaata ttaaagggag ctcacaaaag agcatggttt caccagccct cactaaaaac 54720aaaattatgg gaaaatgctg taaaagaaac cagaattctt ggttgcaaat gatagaaagt 54780gactctgatt tacctaatca gaaaggaatt tttaaaaaag tattaggtag gtcatagttt 54840gacaacaaga cttggaagat aggtggaagc taagggaagc aagacatggc cccaaggttt 54900caacaggagc aatctgctta ggactttgct gcttggacac ttggtgtaat agctgctgcc 54960actatgcctc gaaactggtg actctgctca ttaactcacc tcctctggtg atctctagga 55020ataatctctg actctcctgt accttgtcct cactaggatt cggtatccac ggcaaaaaga 55080tctattaata gttggtatca ggcctgtaca tgtgttaaga gaaagatgag gaaagaagta 55140tctgcttcta atctcttgaa attatctcca aattgaaatg gtattttggt tgcctaacag 55200cctgaagatg acaaatatcc cctacaaatt tctcctattt taccctcttc ctaactatat 55260ctgtaattta aagtttcaca tattcttttg aaaattgttt tcattgttta cccacttttt 55320aagaaaagca aatgggaaca tactaccact gtttggcccc tttcaaaaat tttatatctg 55380aggaatcttc catattgttg tggacatcta cctacctgat tcttttatta actacctttt 55440atttcatttt atgatcatgc tatcattaat aggcccctat gatgaatata taagttgttt 55500ccagtttttt ttttgtcatt gagaacagtt cacatatgta tcttgttgtc ttttccaagt 55560atatctttaa ggcaaattct tagcagtgga gttgctaggt ccattgcgtc tatggtttaa 55620actagtgcct tcaaaaatgg tattttatat actcactgtt taagagtgct tcttccttct 55680atccccacta actttggcaa actgaatagt ttcaaacttt aactttttat agcttgttgg 55740caaaaaatgg tatcttgtta tttgatcgtg tttttttaat tgtgaggttt agcgactttt 55800tgatgtattg gtcttatgta ctttctggtg tgtgtgtatg tatatactga ccctattttc 55860aacttatttt tcttttagtt tgtttgtatt ttccttaatg attttcagga aaccaatttt 55920attctttctt cagacagttg tctaatgttc tgcttctctt gccatttgaa ttttgtgact 55980acaaaattca gatgaaacaa taatagcata aagaacttgg tgggttatct tttgttttgc 56040attattgatt gtttattaag aaatattctt taaaagtcac cttgcttaaa ttagcaagta 56100ggaaatgctt tcaataaaga gaactgtcat gtacccacta ctccttactt actgaatcat 56160cttcttttgg atagagaaga taaaagtgaa aagggaattt aagagttcct gcctttttcc 56220ttgtctttag cattatatag ctgtttaatg tgtgggagtc taatttcttt tttctttctt 56280gagacaaagt ctcactctgt tgcccaggct ggagtgcagt ggcacagtct tggctcactg 56340caacctctgc ctcccaggtt caagcaattc tcctgcctta gcctcctgag tagctgggac 56400tacaggcatg tgccaccatg cccggctaat ttttgtattt ttagtagata tgggacttca 56460ccatgttggc caggctggtc ttgaactcct gacctctagt gatctgcctg ctttggcctc 56520ccaaagtgct gggattacag gcatgagcca ctgcacctgg cctaattttt ttattgttct 56580ttttggtgtg aacattctcc cctcctccaa gccttttgtt tttactattt tcatgttcct 56640ttatatgtgc tgctgttttg tttcatctgt aattatctct catccccttt tttggctatt 56700ataatatata tatgtacgtt ttgaatctga gctttgaagg taaattcact gcagctgtgt 56760tggttgattt tagataattt gtgtatttcc tcctttgtct tttttaaact ggagtcattt 56820gtagttgttt atacagaatt ttagttttta aaaccacaag tctttcatta taggttgagt 56880tatgaattca tagcctgtta tttaaatgaa gcttttgaaa tctgttttac tgatctgtat 56940catatctaac tacgccagta tttccttcct tgtctgacgt gaactctaaa attatgtgaa 57000cactttctcc ctgtttcctg gcatttccac tcaaacttgt tcctcattct tagttagaaa 57060tatatccaga attgtagttt ctttctaatc taatgacaga agcaaattaa tcaagcatgg 57120caagaattta ttggaaaact gcatgtagtt gaaaatatgt ttagtatata ttttgacagc 57180tgtgaagtct ctaattttta ctgtaccttt tctctgttcc aattttatgc tctattctaa 57240ggatgtaccc atttctacta cctgactagg gagcatgtgt attgtatccc agcagatttt 57300ttttttcata gatagatatc ctttagatat ctgttatcca gtgtaggtag ccactagcca 57360catgtagcta tcattatgtt taaatgtaaa taaaataaaa taaatttact gagttgtttt 57420tgctagctac atttcttgtg ctcagtagct acatgtggct tgtgattact gtattaagac 57480agcacagata cagaacattt tcattattgc aaaagttctg ttagacagtg ctgttctata 57540cagtgtcatt ctgcctctca ttctaaaaag ttctaattcc tgaagttgat gtactctttc 57600tgttgctgtc ctctagctta atcaaaataa atttgagtct ttttaaaggt aggttgcatt 57660ttacatactg atatttctaa atcagaggct atttatatta ctttttttat attactttta 57720aaaattagct ttattggagt ataatttaca tgcaataaaa tctacccatt ttaaatgtac 57780ggttcattga cttttgagaa atacacacac acacacacac acacacacac cttcttgtaa 57840acacacacct tcttgtaaac acaacaacca agatttagaa cactcgcttt atgaaaagtt 57900tccctcatgc ccatttgtag tcagtcccca aacctggttt caggcaatct ctgatctgct 57960ttctatatgc tttgcctata ctaggattac atataaatac agtcatatag catgtattcc 58020tttttgtgtc tggcttcttt cttttagtat aatatttttg aaatttatcc ctgttgttac 58080tagtatcaat aatttgttct tttttattgc tgactaatat tacattgtat ggatatgaca 58140tttctttatt agtgtggtgg gcatttgagt tgttttcagt ttgggtctgt tatgaacaaa 58200gctgctgtaa gcattcatgt gcaagacttt tgtggacata tatttttgtt tctgtttatt 58260caataccttt gagtagaatt gttgggtcac atgatgtaga tcagttgaac agagtagatt 58320ccagaaaagt tcacatacac atttcttgac aaaggtgctg agattattca tgggaaaagg 58380ataatctttt aaacaaataa tactggaaca atagagaaaa caaagtgaac cttgactttt 58440atgtcttatc atatacaaaa attaatttga agtggattgt tgacctaaat gtaaaagtaa 58500aatttaaaaa tataaaactt ctagatgaaa acataggaga aaatctctgt gacttttggt 58560ttaaagattt cttagacagt acatataaaa ttaactatat aaggaaaaaa tggacaaatt 58620tgactttatc aacattaaaa atttctgctc attgaaagac tcaaaatgaa aaggcaagca 58680gttttggaga aaatatttgc aatacatata tctgaaaaag gacttgaatg tataatatat 58740acataaagat gctcttacaa cttcataatg agaaaataac cccataaaga gaagggcagg 58800ccgggtgcag tggctcatgc ctataatgcc agcacttttg gaggctgagg tgggtgaatt 58860gcttgagccc aggagtttga gaccagcctg ggcaacatgg tgaaacccag tctctacaaa 58920ataaaaaaat acaagaaatt agctgggcat gatggcatgc acctgtagtc ctagctgttt 58980gggaggctga ggtgggagga tagcttgagc ctgggaggcg gaggctgcag tgagctgtga 59040tcgcaccgct gcacgcttgc ctgagcaaca cagtgagatc ctgtctcaaa acaaacaaac 59100aaaaaaaaaa acaaaaaatg gaaacagaaa ttttacaaaa gaagatatat agatggccag 59160taggcatatg aaaagatgtt taaaatcagt catcagggaa atgaaaattt aaacgtaatg 59220agatagctca tatttactgg aatggctcaa aaagggctta caggaattgg caaagacata 59280gattaactgg aactcttatg catgttggtt agagcacaaa atgatatgat ttcttgggag 59340aaatatttgg cagtttttaa gattattttt gatagccttc tgaatttctt agtgagttat 59400aggtcagttc tgccactgtt tctttctttt ctttctttct ttctttcctt ccttcccttc 59460cttcccttcc ttgcctgtct tgcctgcctt ccttgccttg cctgccttgc cttcctttct 59520tcctttcttc cctttctttt ctttcttttc tttttttttt taaaggagtc tcgttttgtt 59580gcccaggctg gagtgcagtg gcacgatctt ggctcactgc aacctccacc tcccgggttc 59640aagcaattct ccctgcctca gcttccccaa tagctgggat tacaggcgcg ttccaccata 59700cttggctaat ttttttaatt ttggcagagg cagggtttca ctgtgttggc caggctagtc 59760tcgaacacct gacctcaagt gatctgcccg ccttggcctc ccagagtact gggattacag 59820gtgtgagcca ctgcgcctgg cctggcactg tttatttctt ttccctccag ttttatacct 59880atttagagag attagatttt cttgagtact aggaatcact atttttgagc agaattattc 59940aaaactgtta ttattttttc tttaacttga ggcaatgtag gagaaagcag tactgtgcag 60000gtgaaagtta caaacaagaa cattttaaac aagatagtta ctttccatgt attggatacg 60060taacagaatt aattctaata accatcctga agatggtcag gaggcattag ttaagaattg 60120aaatgtttgg agcttgcctg tgttgatggg attaaggcag ggatgattta tgtgtaaatt 60180tatgcgttag taacagcagt aaccgctgta gttacactag

ggttctaaga gcaaatgttg 60240attaaacatg aatgtagcag gagtgataag gtttggctct gtgtccccac ccaaatctca 60300tgtggagttg tgatcctcag tgttggagga ggggcttggt aggaggtgat tggatcatgg 60360gagtggtttg taatggtttt agcactatca ccctagagct gtctcgcgaa agagttctcc 60420tgagatctgc ttgtatataa gtgtgtagca cctcccctct ttgctctctc tcttcctcct 60480actcctgccg cggggacgtg cttgctttcc cttggccttc tgccatgatt gtaagtttcc 60540tgaggcctct gattaaacct ttcttcttct aaaagattac ccagtctcag gtagttcttt 60600atagcagtgt gagaatggac taatacaagg ggaaatatat atggttacca aatagcgaat 60660tagccatggg aaaaagtagc aaataaataa ttattttact ttttcagatg ctaatttttc 60720ttttcgttta ttttaggatt ggtgggagct gtccaatgtc cttaggctgt tttccaaatg 60780agataccaaa agctagttct ccatcgggtt tctcaggctg ctagaagcat tcattattat 60840ggttgtcatt acttcgagtt ctgttgccgc tatgcccaca gtagtatttg ttacataaca 60900ggtgcttgat aaatatttgc taaatgaatt tttggaaaat acaatctgcc acacctttct 60960tctacagttt acaatcttct gttgagatca tccgatagat tttttttctt agatattgta 61020cttttgaggc ctcaaattgc tgtcttttgt attttctatg tctgcagaga ctttccatct 61080ttcactcatt gtattcattg ttttttaaca tctttgtaca tatttatagt aactgtttta 61140aagtcactgt ctgttaattc aaacatctgg ttcatcttgg agtctgattc tattgcctgc 61200tcttttttct ttgtaatagg tcatgttttt ctgctttgcc tgtctagtaa attttaatcg 61260tatgttgaaa tgtagggagt ttggattgtt acttccttta agggtgctga gtttcatttt 61320gtcaggcatt taaattgata gttgatttag tattgtcagg tttggttctc tttgttaaag 61380caggcatttt tcagatttgt cttttgtcct agggcatggt ttttaacttc aaggttgccc 61440tttccaatgt ctcagctaag tatctggggt gttccatgag gtctcttcca ctttgcctag 61500gccagaactc cagcttctcc cagtattata tttcgttacc tctggcgtca tctccgttat 61560gctttcagat cctgcgcata gacagcccag cccccagcca aggacctgag atgaaatcca 61620tacaaaattc ttagtccctt gctccacaaa ctccaacagc cttagcagtc taatctcttc 61680ctgtttacct cagtgaaatc tgtgttccac ttgagttcca tttccttctg tatcagagaa 61740gagccaccat gctgaaagca aggggcacta tgtttctttg ttcttaagga tggtagccta 61800tctgcaacaa ctgtagtgtg atataaaaat atataattta tgttgctgac agttacaaat 61860actgcttgca gtactttgta acataatttt tcagattcaa gttcatatac tctttttttc 61920cacatcacca cacacatatt ttcagacttc ctcctcatcc ttcttcttgc cagtagttgt 61980attataattc ctgccagtag ttacattata attttggtta tatcaatatt gagtttttat 62040gggattataa ctagataaat gccattcata gttaagtgat agagtatatt gtgacttttt 62100tcctgcatgt tttatttttt ctggacttca cagttgtctc tctttttttt aaaaaaatta 62160gttttcaatg ttcttagctt taattcataa actcacccct aattgtataa atctctcaac 62220atgtttaagc acatttggca ttatatcaat tttatctttt ccaggtgcct tctaatctgt 62280cccagtctgg actaattgtt cttcctggct tgctgtatgg ctgtctactc aagatgtccc 62340ttcaccatca ttctagggat tcccttttcc tctcttgtgg gttagattct tcagttcttg 62400gagactgtca tcttctttca tggtttccca ctcttgtttt ggcggagcac atctttagta 62460acttcctgac aaagtgtatg gtttgagatt tcgctgattt taaaatgccc ttattatata 62520gtcacacttg atttatagtc tgtcttggta tagaattcta ggctgagaag agttttccct 62580caaaatcaga aggttttgcc caattgtttt ttagctgcta gtattgctgt taaaaaggat 62640aatgtcattt tgattctaga ttcttttatg aaacctgttt cttctctggc agcttttagg 62700atcttctgtt tctttggtat tcagaaattt catgagatat gtgtgcttct attttggtct 62760tattttcatc tgttttgcca ggtactcatg caactttcca gtttgaaaac tcacatcctt 62820cacttttgag tatttttctt gagttatgtc ttcggtttct tctcagtgtt ctctgtttct 62880ggaactccta aaatatattt aacatcctga actcttagtt tttgtttagt cttctgattt 62940tcatttgtct ttttattctg tatattctgt taattcctcg gcttcatggt cttctagccc 63000ttcttttgcc tatcttatgg ggttgaggat taaacagttt atatacctga ggtgcttagg 63060atatgtctgt catatagtaa gtgcttgtgt tagctgtaat tgttgtttac tttcataact 63120gtcttgaggg aaaggtcttt ggtcttgatt ctttgacttc ttggctgtac atgaccttgg 63180acgagttatg taatctcttt gagacctacc tccctcttct gtatagtgtt aataagctct 63240agctctcaga tgtttgtgag ggtcgaatgg agtatatatg tgaaaatgtt taataccttt 63300gtacagaatt aatagttagt acgtggatct ttcaaatatc aaaagttttc agtttgatgg 63360gaaaatgatg tctgaatttt cagggttatt tttaagagta cttgattatg actgtcttgt 63420aaatctctat gagctaggta tacttgcact aaatgctaat gctttttaaa gaagttatgt 63480cttaatattc agtctcatta tgttaggttg aagatagaag attatgaaaa tattctctga 63540aaagctctgg ttttacttca gattgtataa atctgtgtaa tgtaataatt atttaagaat 63600gacatgatta ctactctaaa cccatagaag gggtatttgt tggattattt attttcactt 63660aaatggtatt tgagattagg aaaaagaaaa tctgtctttt ggtttttctt gatagtatta 63720atgtaatttc aaatgttagc tcatttttgt taatggtggc tttttgtttg tttgttttgt 63780tttaaggttt ttggattcaa agcataaaaa ccattacaag atatacaatc tgtaagtatg 63840ttttcttatt tgtatgcttg caaatatctt ctaaaacaac tattaagtga aagttatctg 63900cttgttagag tgaggtagag ttaaagatac attttaacag aattgtattc ctaaaccgat 63960taagtcaaga agtccaagag cattgttaga tcatttagaa agtgtagtga tgaggtaaaa 64020cattgttggc acagattcat gttacttgat ctgctttaaa tgacttggca tctagcccat 64080atttgagccc ataaccgtgt ggtaatttga agtgtaattc acagtagagc ttctgttaaa 64140gcactaatag catcttccat ggaggtatac ttcagagtga atataatttt gtttatcctg 64200tgtctctaga gctattgact gaaaaagctg ttagggcatt ctctaactgt acatcaccta 64260agttatttaa aattgctgaa ttaggtggct tgtcttgtct aggacagagt tttaaggact 64320gcccacctga ttgatagagc tagttgacct tatctttaac tttttgtttt tcttttgact 64380ttgggagtag agatgtgaaa aggtaaaaag gaaggaagga agagaaaact taactctttt 64440tgcccatgaa gactgttttt ccttctcaaa atattgacta ttttctgatt tgtaaaaatc 64500ggcacataaa acgtgttatt ttttacttga cttttatctt tcccatgtga tatctataaa 64560ttatagatag gaaaaattta tctgtaattt agtgatcttt ctagtgtgat aaaacgtcag 64620aagtactgag agtggagtgg acattgatat tgttactctc agtaagtttt cactgatttt 64680tctcagagtc atgaaggaac aaacgtttgt taagtcctta tcacttatta gataacacaa 64740aacatgttgg gggggtgtgt acagaggtga gtaagatgta gctcccattc tcaagtcgct 64800tacattctaa tgtaaaaggt agacaaagca ttacagaaga agtaactctg ctatagaagg 64860ttgcaatgaa gagaacattg gaaacactaa ttttacctta taaagaaggt ttcataaagg 64920aaggcaagtt tgagctgggg tgaaaaggac cagtaagggt tgactttcaa gccaaggaga 64980ggaggggaag tgatgttaca ggccaaagga atggcattgt aagaagcttg ttggcataaa 65040agtgtttaga atatggcagc gaattcatta tcatcagatt gtggtgtctg tatgttgggg 65100gtgggagaga attgtggtgg caataggcaa caagataaaa gaaagtaaaa ggtgttatgg 65160aaacttaatg ggtccagctt acaaatgatc tatgcattta ggggtctttc tcttttcctg 65220ataaacctct cctacaaaga gccttgttgc ggataccata gtgtttcttt ggaggaaaat 65280aaaaactaca aagctttgta ttttttgcac aactggattc agaatataag taataaaaaa 65340ggacaagaac tttcaaaagc tagaagccat taaactgagt cacttcaggg ttagactatc 65400agaactgggg atttagaaag tctcagaatg gaaatcgaag gacaccaaag acaaattcgg 65460cctttttcaa aattttattc tagtttaaca tattcaaaga aagggaagga aattcttttc 65520attcctgtgt gtagtgactt cctgctttaa gaacttagga cttcagctgt actatcagta 65580ttgtaggcca cttaacatta ttatggttaa agttggcatt ggagagagcc taggaaccta 65640actgcctgtt tgtttttata tttccaacca ttggattccc aagttaatga agtctgttta 65700ttagttgagg gtagctctta atgcatatat tttaatgccc cttccccaca tggaatcata 65760agctttcaga actggagagt acctgaaaga gatcatttag tccaaccttc tcattttaca 65820gatggggaat ctgaggccta gagaagttaa gtgagttgaa caaggtcaca caggtacata 65880tggtagccga ccatccactg tttatgccaa tattcccttt acgttttgct tttttgcttg 65940ttcgttttaa cctctccaaa ttttactgac ttcagaagtt tctagaacta agttatagca 66000tgttttgagt tctaatgtca ctttccgatc ttctttacct tttttctacc tctgtttgta 66060tttctggttc tggttaagtg agtctggtaa gcagcaggtg ttctatttta tttcttttat 66120ttttaggata gtattacatg tgatatatat gtctttgcaa acatacataa tttgaagatc 66180ttaaaatatt tgcactaggc atacccacat ttaatagtat gttaaatctt ttatagcaat 66240tatgatatac atgggtgaag aagagttcct aatatggcct ttctgattaa ctgtatctgt 66300ttatatctgt gttttcttca ggcattcata acattaagca aattcaggtg tactgttact 66360taattgaatt aatcagtttg ttttgtacaa gtatatttta tttttgttcc ttgttgtata 66420atctggtagg aatggggaag gggagatagt gaataaagag atgtatactt cttgcctttg 66480aggaatttaa gttttcactg tataccaatt ttttaaaggt atttactata tttcagtgca 66540tattttattt gacatacttt atcattttgt ggtaaacctt tagctttact aattttcatc 66600tattaagttt tcttttgtaa gatggtgata gcttcatcaa agagagtaaa gaagagacct 66660gcctacctag ctgattctat ggcaaatctc acttctctgg aagcttttcc tgttaatctt 66720attccttcag tttctgcctc ttgtttcata aaaactcatt ctttaaatgc ttattcattt 66780ctcttgtctc atataaacca atatgaggta ctggtatctt ttgagtttta gttataagga 66840agcataaatg gttaaattta aatggctaaa ccccatttgc catttgtgta tctttaattt 66900tagtttgttg agagacttat cactaccaaa ccacaaagaa tttaaaagaa actgtcagta 66960ggtataggtg gaaggagggc atttatcaga gattttaatt taagaagaaa gtcttcatcc 67020ttatcctacc aacccccatt ccctgagcat atttatcatt actagtccca gcatatttgc 67080tcccatattt cctatgctta cctgtgaaga ttttcataac tttttccttg ctttttactg 67140tcactgttgg ttctgtgatt tatgacagat actgctcttg taggaatgct ggctttgact 67200gaaatttgtt actgcttttg tatttaaaac ttttttttta ttataagtag aattatggaa 67260cagtagtaga aaaagtttga cttttgtaat cagagatact gagcttgagt tctggctctt 67320tcatttgtat actgttattt ggggcaagtt ttttaatgct cttaagtctt agctttctca 67380tatataaaat ggagataata acagttatca cgtgattgtg aggatgaaac aaaaaaaagt 67440ggaaactctt tgtaaggtgt gttcatctgg ttgacactta gtagtcatta cttccacttt 67500ccgtccatat agtcctctta acagtaatat ttgagaggca tttttattaa agcagtctta 67560aggagtgttc gtcaaaccac atgttctggg atcctgagaa agtaggggaa gtttagagaa 67620ctgaagctgc acaaaactaa tgtttatttt ctgttgtgtt gtcctgagac cagcttctta 67680gattgtgttt cctagtccta catctctgat tccttataaa atattccatt atgaattctt 67740cactattgac aatttctccc cttttatctt aaaagtacca aagaaagtgt aaaatgtgac 67800tgtcttgtca gtcctctttt tcctgttttt catgtcagtg ggtatgaaat tactagcaag 67860gatgcatata tgtgcatatg tcattactaa atgcattttc tttctagaaa aactcaatat 67920actaaattgt actaaaaagg aaaagcttgt tttgttttga gtggtagtat gaaagttgtt 67980ttattttagg tctgaccagt tagaaaccaa tggattgtag tttatttata attagttaaa 68040ccttcatgtg aatttggttt tgaattacct ttaaggtaga gaagaaacta tatagatgtt 68100tttcagggtt tctaaatgta caatacaggt tcacaatcac ttatttgaaa ctcttggggc 68160caagtatgtt tccattttca gaaattttag ttttcaaaag gtagcacaga taaatatact 68220tttacataaa caccccagtg gggtgtgggt cagtacctga aatgaaatgt tttactcttc 68280gctctaagtg tattaaatat tatgtacaat cttattactt cagatcagga tttgctgtag 68340ttgagtttgc cataaaactt aagagaaaat tttagatgtt ttgaactttt gggatattga 68400aattgcaggt taaggagcta tggaccttta tttgttttaa aatgctaaga gtttatttta 68460agtaattttt aaaaaatttg ttttgcatag tagttggagt taccagggta ctgctaacca 68520cactgatatg taagatctct ttctgagcct tttattgttt gtaaacatgg cctgttaatc 68580attagaaagc cagtacatac taacatatca ctgctattaa 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gatacatatt taagaattat 69480ctttaaaaat ttcaaaaatt ttaattttac tgttgtgttt taggaaaaag tattgcataa 69540agctattaat attgtcagga agactaaagt gcagcataga ctaagaatta ggaaaattcc 69600tagactaaaa atagtataag gagagggttt acctactatt tgaggcagtt ggtctaatag 69660taagcaatca cagggagaaa gcagaactac ttaactcttc tgtgttgagg aatgacataa 69720aaggtaggaa aggatataac aaatgttgat aagaggagtc tgatggatga gaggagggaa 69780ctgctttaaa tgagtttcta cttcagacat aagttaattc tcagagccca caaaaacttt 69840cacttttatt tgtgaaatac aactcagttc tcatggctta acactttaaa ccatgagaaa 69900actgaagagt tgagaagctt ggcagatgct gctgtgatag tcaaaaagaa agtgggtgcc 69960atgagctact attgatgtat ttgccattga tccctcctga aaatctagaa tggactttca 70020gacaaatggt ttgaaaattc taaatcacta atgattgaga tttagtatag gtttactaag 70080aacgggtttt ttttgttttt gtttttggtg gatttaggct gttgcttact aagcaaagca 70140ggctttagtt gaggtttatc ttgctttaaa cagatattta acagattttc ctggaggttt 70200ttgtgtacca ctgggaaaat gaagttaggc agatgactaa gtgaaagctg tcctgctgac 70260tccttataat gatagtcatt gtctaccaga agatctctcc 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ctgaagcttc caaaaagatt 74520ccaaaatggg aatgtttcct tgattgtgtc accatgcttg catttgatga aaacttgtag 74580ccggctatac tgagaaatca tatctgaaga aaggtggtac ttccaatctt tttgtgacct 74640actttattat tgttttttta atgtcagggt tttttttgga atggagaaaa gtatttgata 74700gaggtattgc aacagtctta ttcttcttca tgctacaagt atatttgact ctttctaaga 74760tacttgcctt cactgttcaa ctgtgtgact ttttgtttgt ttagcattac aatcaatatc 74820ctagtaggat gatttaatca atgattttta attggaacaa atagtttttg taatggtcta 74880ggcttttcca acttaactgt gctctcacat gtggtctctt tttctccctc tttcctcctt 74940cttatacact ctcacccaca cacatatgca tacataccct gtctgatgta tctgcttctt 75000cagaatagtt ggctgtgctc tgctgatgat gagaacttgc catttaagaa ggacttggga 75060tagtccatgt catcatgttc agggataaaa gtaaaaccca agggcattta aactttattg 75120tattttattt tctgtttcca gtccaaatta aatccaagag aaggctccat aatcaaaaag 75180taaggacata ttttaaattt gccaatggga agatattcta gtcattacag tctggtaata 75240ctatcaattc tgtttctctt cagaggtgag gggagactat

ttgatgaaat cgtaagtcct 75300gtagggtgtt gtgaaatagg gccagaatga aagatagcaa gaatagtgtt atgaaaataa 75360aatgcaaagt ttataatatc atgtggtaaa atgtaatagt atttacttca tcagtagaac 75420tgctctagta gctgtatatt ctccatcctt gcataggttg gaatatcccc caagtgaaaa 75480gagattgatg ggctaatagt taatagaaaa tggagatctg tacatacagt gttaagaatg 75540tagatattaa aattgttata tttagctgtt acataatatt aagactcaga gttaagtaat 75600ttcactgaaa ttgattgctt tttgtgtctt ggagtcaaaa taaataactg aaatctacta 75660tacttggctc atgcttaatt aatatactta gaccatattt cggatgaatt attcacagaa 75720tctaaaggag tatcctcgtg ttcttacctt ctttatccct gtgtttattt aaaaaggcaa 75780aaaaaatgga gcagatgctg ttggttgacc atattttact gaacagtagc atttgtgttt 75840aggttgaaac agcattagaa aactagatac ggattaaagt cagtggtagg tttttttttt 75900tttttcttcc aggaatgttt cttatagatg atcaaacagg cacaggaagg ggaagtgttg 75960tgatcaatat tatccagtta atattagcat tcagaggaaa atttgagtcc tctgatacac 76020tgttaaattt ctttctatac tatcaagtcc acaaatcctg gaactgcaaa agaattttga 76080gactgttcaa aataattaat ctctgtatag gctcaggctt 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cattaggtta gtaatgccca 76980aataatactt tgggttttca gtgttgcaga gaagtcagac aactgatagt tattataaag 77040aaaaatgttc tgagagtgag gtaaccgctt aagggaagga agcctccttc tgtcttattc 77100actaatttac aagaagataa ttgtgttaca cttccttagg agtcattcat ttgtatattt 77160gacacttttg ctttatgaac atgtgaagat tattcaaaag taagctgttg gtgatttttt 77220tcttccaaga aagcatgcca cagggcaact tctagggttg gttctcatct agtcctgtgc 77280tccacactat ctgcatctgc acttaagttt caatattaga taactcacat gtttaaacta 77340tgaagaaaga gttaaaacat cctgagaatg ctagtaagta tgtatttttg aaaggacttc 77400caaaatttga gtttaaagag gtaaactcct tttacatgac aaagttactt agaaacacta 77460ctgctgtttc cctctccctt gccttctccc tgtcccatgc atacccccag ctgtgttcca 77520gaatgatggc acataaagta aacattcata tttatttccc tttttttgtt tttttttttt 77580tttttttttt ttgcttgttt gttttgtttt tttgtttgag acagtctcac tcaatcaccc 77640aggctggagt gcagtggcaa catctcagct cactgcaacc tctacctcct gagttcaagc 77700gattctcctg cctcagcctc ccgagcagct gggattacag gcgcctgccc ccacgcctgg 77760ctaatttttg tatttttagt agagatgggg tttcgccatg 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atgacagaat ctcagtatca 80340gggtagcatt taaagcaaaa agcattaaac tggatcaaaa atgtcatttt acatcaacac 80400agggtacact ccaggtaaag acttaacagt tatgaacgaa tgtgctaaac atcaaaatgt 80460attaagtaaa agctgaaaga aatgaagaat aattggtaga aaagcaatga taggaatcct 80520taatgtactt caaaaggcac gaagtaagaa ggtacagtca tgtaccacat aatgatgttt 80580gagtcaacac ctgacctggt atacgacaat ggtcctataa aattataatg gagctaaaaa 80640gcttctatca cctagtgatg ttgaagccgt tgcaatgcaa tgtatcactc acatgtttgt 80700ggtaatgctg gtgtaagcaa acttactgta ctgtcagttg tataaaagca tagcacagtt 80760atgttcagca cataatactt tataatgata aacgaatatg ttactagttt acgtgtttac 80820agtactatta ttttagcatg tgcttctgct tattaaaaaa tgttaactat aaaataatct 80880ttaggcagat cctacaggag gtattccaga taaaggcatt attgtcatag gagatggcag 80940ctccatggat gttattgccc cttaagacct tccagtggga caagatgtat aggtggaaga 81000cagtgatatt gatgatcctg accttgtaga ggccaaggct aaagtatgta tttattagtt 81060tttaacaaga gtttaaaaag taaaaaaaaa aaataaattt aaaaatagaa aaaatcttaa 81120taaggataca aagaaaaaat gtttttgtgt agctgtgtaa 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gaatcaagat ctggattttg 82860gcaactgaat agatgcgtag gaatcaaaga tgagtatact aaatgaactt aatctatgat 82920ttgtctgtca ttttattgtg taccattagt gtgtatgcat gtatgtatgt gccaggtagt 82980taataggctg actgtgtctc ttagctccac tctgctgcct gggcctttgc catagtgctg 83040gagttccctc acttctcttt tctgtaaccc tattatatta ctgctgtctc tcagctgtgt 83100ttcattcctc aagcagaaag aagatgggag gatcatatag tagttgacta gaagctgtgg 83160agtttgagtg ctgggattat atctagttcc attacttatg aagattatat ctagttccat 83220tacttcacct catctataaa atggtttacc aatagtacct accttacatg gttttatgag 83280tattaaatta tgtatttcta aagacattta gaacaataca aagaatatag tgtgggctca 83340ataagtgacg atggtgttag ttattagaag gccatcgagt gctggagaaa ataattgaat 83400atcattgatg gaaataaagg aagttttcca cgttaaaaag cttcggtttt tggaaatgtg 83460tgttttcagt atttctgaga ttaccaggta gaaattccag ccagatgttg gaattctgca 83520ctggcagttg ggaataaagt catgttaaag gagataagtt ttggagttat tgtgtaaaat 83580tggtaaagca ctggaataga ttggtttgac agaggggaaa ctctttttga gataggccta 83640tatttaggga caagagaaga gacaaccaat aagggttgaa 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ccttccctac tgaaaataca 87060aaagttagcc aggcgcagtg gccatcccta cagtgctagc tactcgggag gctgaggcag 87120gagaattgct tgaacccggg aggcaaggtt gcggtgagct gagatcgcac cactgcactc 87180cagactgggt gacagagcga gactccatct caaaaaaaaa aaaaaaaaaa aaagatttga 87240aaacagagta ttttttaatc tgcaagagct ttacagcctt ttattcatat gtataagctt 87300ttaaagatga ctaaaatttt agtgtggact ttccactcat tggaaatcct atattgcagg 87360tgttaattca attttagtga gtgtgcatca tggctcagag agataggact agaatgagga 87420ggtcacattg gagactctga aacagataca tgtgagcctc ccaacttttt aatatttgtt 87480aatctagaag tgttgaattt tgggtgctga caaggcagca ggtagataag aattgcaaag 87540ttaagaaaat agactgtaat attgatggta aactaattga ttaaatttta aaatgtactt 87600ttccatgttt ttctttgaat tgtcagtaat tttgtttcaa ctggtattca tacatagatt 87660attcacccaa tgttgacaac tagtagattt atatattttt tatgttgcct atcctttttt 87720gggtaaggat taacagaatg tataatcacc tacattatag gtacactact aatcacttgc 87780tacttgaaaa aacctaaagc tttgaaatct ttttattatt gcacacaaac ttatgccaaa 87840aatggagata aagagaaaaa tgtcatccac taaaccccaa caaataatgt tgacaatgtg 87900gtctactcgt agactcgcat tgacttaatt tttttaaatc ttattgcata ttttgactag 87960ataataaatg catatggtta aaaaattcac atggttcaaa aaagtacacc tcccactcat 88020cttccatgtg atatttcctt tctgcttagc aattctgtat ttatcttgct aaacatgaat 88080gacagttgtt tgctgaaatt acattaaatg tgacgtaata aaatcattgt aagtttacat 88140tttttaactt taataatttt taatgtttta atgaagagta tgaagagtag tagtactgct 88200cttcaaagta ctactacttt accttacctt ttactgtttt gttaagaaaa ttaggccggg 88260cgcagtggct cacgcctgta atcccagcac tttgggaggc cgagacgggc ggatcacgag 88320gtcaggagat cgagaccatc ctggctaaca cggtgaaacc ccgtctctac taaaaataca 88380aaaattagcc gggcatggtg gtgcgcgcct gtagtcccag ctacacggga ggctgaggca 88440ggagaatggc gtgaacccgg gaggcagagc ttgcagtgag ccgagatcgc accactgcac 88500tccagcctgg gcgacagagc gaaactccgt ctcaaaaaaa aaaaaaaaaa aaagaaaaaa 88560gaaaattata tagaaataaa attccagcta ttccaaaact gcaccttgaa tacaggtaca 88620gaattgctaa aaccgtgtac cattttgtag ttttagcatg cttttgtgta actgcatctg 88680gtgtttgatc ctcatgagag ccctgttaag gaagggtaca tattattgtc ctcattttcc 88740ttcgaaaaca catcagagtt tgtattttga ctgtcagcat tcaaatacaa gtcttttatt 88800tataaaattt tggtctttat actgtggcta aaaatcttaa atcacttgtc atgatttgaa 88860atggtttata ccgatttttt ttgacattta tacacacata cacatatttt taaattgtct 88920ataataaaat catgctcatc tttgaaaaaa tattaggagt actacagtgg atacctacat 88980acttgctatt cagcatacct ggttttttgt ttgttttttg agacagtctt ctctgtggtc 89040caggttggag tgcagtggca cgatctcagc tcattgcaac ctccgtctcc caggctcaag 89100tgattgtcct gcctcagcct cccaagtagc tgggactgca ggtacacatc accacgccca 89160gctaattttt tgtatttttg gtagagacgg ggtttcacca tgttggccag gctggtctcc 89220aactcctgac ctcaagtgat cagcccacct cggcctccca gagtgctggg attacaggtt 89280gtgagccact gcacctggcc tgtttttaaa ttcacataaa tatgttttat atttttcatt 89340agggagaaga aggttgtgtc tacaattttt aagacattgg ggagatttag atgccagtag 89400taacttaaaa gagaaataat tgcaaattct ttttcctctt gagtatactt tcatttaagg 89460tacagtgttc tgtaagttac ttttaccgtt aaacttctta atgttgctta ttgtttgtct 89520tacattttta ggttggattt ttcttaagtc acatgtctaa taaaaaaaac ccttaaatac 89580ctcatttatt cgtcttcgtt agtgaatgca ttgttgtaca tattagattt ttctctttag 89640ataactcagc ttcccctatt aagtgccaca tgtattacaa aattttattt atgttttatt 89700gtttaataaa ctcttgagaa ctagatacat tttaatcatt tgtaatactt acattttcta 89760aaacacttca tttttccctg gtttcttcaa caaagagatg catgtagtac aaggatagct 89820ttacctgtgt tagaagattg tttcacacat ttacatcaac tgcatagtcc tgtttttgtt 89880gggccctaat gccagcatca ctttttgcta ctgctgtttc tgccttaaag gcaatatgcc 89940tctgtctagt ttgctgattc tgatactctt tcccctggaa agtaggtaat caagtttgtg 90000aggagctgtg tgtttaagga gtccataaat ccttgtgggg agccctaggt gtatagagca 90060tagctgtagg gcagaggcct ttgacactta ttctggatat gcagtggcct ttgcctatgg 90120ggttcatggg tcagagcgct gttgtgacct ttgaataaat gggttgttat gataattgtt 90180ttaagggagg agagttattc tgatatcctt tgtattgata ttgctcttat ttattattga 90240gctggattta agtattaatc atttaaggtc aaatttctaa tgtatatatg ttcttaaatg 90300gctacgaccc agttaccata gcaatttagt gaaataacta

taatggaaca ttttttttca 90360atttggcttc tctttttttt ctgtccacca gggagtaact attcccagtc agaggcgcta 90420tgtgtattat tatagctacc tgttaaagaa tcatctggat tatagaccag tggcactgtt 90480gtttcacaag atgatgtttg aaactattcc aatgttcagt ggcggaactt gcagtaagtg 90540cttgaaattc tcatccttcc atgtattgga acagttttct taaccatatc tagaagttta 90600cataaaaatt tagaaagaaa tttaccacat ttgaaattta tgcaggagac tatatttctg 90660aagcatttga acaaattaat tagctttgtt gttcaactca ttgggctaaa gaagccaaaa 90720gcaatgggtt ttaatgtagt cgaagccaaa ttatatttat gaaagaaata ttctgtgtta 90780taaccaccaa atacagccca attctgacta gatgatggaa gaacctgtcc catcagaggt 90840ccagcatgag gtccagcaga ggtccaccag aggagttcag caatttgctg ctcttagggc 90900agggatcaat tccttaatat cttaggaaga ctaggtattg acagtaatgg tgacaaagca 90960atgaaaagga aaggaagaag tgataagacg tggcagcaag ctgaagtatg atgagtaaag 91020aataggaatc aaagtatgtg gagtgttaga gaaaacctgg atttagatcc agattctagt 91080cctatctctg tcattaatct attgcgtaac cctgagcata tcatctacct ctctttgagt 91140ttgcttgtca ataaaatgaa gagactttga aatctgagac ttcctggata agtactaaat 91200acagattatg tcactgatgt ctgcctctat ttatttctcc cttttaccct aatctctata 91260agtctacctc agtcatcctg atcctattct acttctctga tgttgttgtc agataggtgt 91320gatcatcctc atcagatctt ttctgtattc ttagagacag ataactttat caaagaccac 91380agatttatta gtatagcatg ttaaagtctt ctaaagagtc tcattgatgc tcttttcatc 91440tcagtacaat ttttaaaact gctgaatgca aggtactgag ctgttggaag tgactgacag 91500atgaatgtaa cagattcata gagaaggaaa aaggaagaaa aactcatgct cttcctatag 91560tattgatatc agtgtaagag ccaagagaaa ggtataaagt atcatgcaga tattaaggga 91620aagaaaacat tcactttagt aatctttcct cattttctag tttcctctta tgtactatga 91680tttaatactg tagtaaagtt ttaataaaat atgagctata tgtaattaag tgggaggttg 91740tggggctagg cacgaggctc acacgtgtaa ccccagcact ttgggaggct gaggcaggcg 91800gatcgcttga tctcaggagt tcgagaccag cctggacaac aaggtgaaac cccatctcta 91860ctaaaaacac aaaaattagc tgggcatagt ggcacacacc tgtagtccca gcttcttggg 91920aggctgaggc aggagaatcg cttgaatcca ggaggcagag gttgcagtta gccgagatca 91980tgccactgca ctgcagcctg gacatcggag caagactttg tcttagaaat aaataaataa 92040atataaaata aaataaatgg gaagttgtgt atataaatta taaatgctac attcagaaaa 92100gcttttgaag gttgtcagac agtttcttaa aggaagttca ccagttcttt attgaacatt 92160gaagaaaaca tacagtttag actggcatta aaactgaaag aagtggccag acgcagtggt 92220agacgcagtg gttcacgcct gtaatcccag cactttggga ggtcaaggtg gatggatcac 92280ctgaggtcag gagtttgaga tcaggctggc cgacatggtg aaaccctgtc tctactaaaa 92340atacaaaaat tagccaggca tggtgatgcg tgcctgtagt cccagctact tgggaggctg 92400aggcaggaga attgcttgaa gccgaaggtg gaggttgcag tgagccgaga ttgcgtcatt 92460gcactccagc cagggcggta agagtgaggc tccgtcttaa aaaaaaaata agtaaataaa 92520ttaaaaacta ctgaaagaag tattacaggc aatgggaaat agcttgagtg gaagtgcagc 92580agaaggaaaa agctggacaa gaatgtagtg tcagagaata ggtatggaac gtgtgagtga 92640ctgttagagg atcttgaatg gggataacag acttgatttc ataaatactg agatgtcatg 92700ataatacttg aggactaaac catgttttaa ggacagttgt atgcaaagtt gtaatcgcaa 92760gaggaaaaaa tagtggaaaa gaaaccagta ataaaacttg ccttaatgca ggtatgctaa 92820gacaatcaaa tgggatttca ttaatttttt atttgccatt tatagccaaa gattttgtaa 92880aagttttgag cccagtcagg tgaaatagtc tcagaaagaa agaaaagtga atctgagact 92940tggagacatt aatgttgata ttttggtttt aaacgtgttt taaatccggt aaaagtgagc 93000ttctcacatg acaatattca gtgggtactt gggagtatgg gttcgaatct aggtaggaga 93060tatatcgata ttttgggcat catcaggaaa gggagagtag ttaagccttt catataaata 93120atggtgtggc gtttgggcat gggaagtctt ggaggaaagg aagaaaagga gagggtgagg 93180actgagataa gaatggcaac ttgggtttag gaagaagaag aggaatcaat gtagagaaca 93240gatagtgctg aaaaatacag catcttctgt agggattggc agctttttct tgatttttgt 93300cttaatattt ctaagagatg gaaaaagcta ctatattcta gacatttaac agggttaaaa 93360atgttactaa aagatgatca atgtggtttt cattcaagac tataacaata tgtatatatc 93420caaggaaatt taattctgac ttaaaaaaat tgttttgctt gtatagattt agggacacaa 93480gtgtaatttt gttacatgca tagagtgtat agtttcaagt cagggctttt aggttgtcca 93540tcatctgaat aatatacatt gtacccatta agtaatttct catcttctac tcaccgtttc 93600aagtctccac tatttatcat tccattcttt acattctgat tttcatttac taggtgtatt 93660agtctgtttt tgcattgctt taaagaaata cctgagactg agtattaaac aggtttaacc 93720tgttttcttt atgaattctt ctttaattgg ctcatggttc tgcacgctgt acagaaagca 93780tagcagcatc tgcttctggg gaggcctcag gaagcctcca atcatggctg aaggcaaaag 93840gggagcatgg tgagaatggg agcaagaaag agaaggagtg gtggggagaa ggtgccaccc 93900acttttaaat gccagctcac ttaccaccaa gaggatggcc caagccattc atgtgggatc 93960tgcccccatg attcaagctt cttccaccag gccccacctc tagcactggg gattacaatt 94020caacctgaga tttgggaggg aaagatatcc aaactatatc actaggtctg gatcttgtta 94080tttatttttt ggaacatagt catatatatc caaggatata tattgtagaa gtccacagaa 94140ccatactaat attggacttc tgcttagtta ggtcttatct atctgaaaca tgatattcat 94200attgcagaga agattatttt ctttagtgat tgaggaaatc tttactactt atacattttt 94260aatataatac tataatattt gaagatgcac attttagatg tagtttaatt gaaacctgga 94320aatactatta atttgctttt taaagtccta aaatcaggat tatcagattc tgaattaatg 94380gagtttaaat caaaaagatt acaaggcagt ttttcagttt tattctggtt aattttatca 94440cagctttgga atcctacttt gtttatttgc ttcttgaagt tagatttccc agtgaaattt 94500cagtatcaca taaagtctta tgaaatggct cattgcactt tgaactttga gtcaaggaag 94560tgaaatttat tgatagattg ttggtgtaat atttatcctg tttgtggtag cttttttgaa 94620taataagtgt cttagaagac catgttggag tagcctgcat gcttttatca aacatattaa 94680ttatgtgatg gctgatactg ctttagatat tacatagaaa tagtagtagg tgtttactaa 94740actggaaatt tcatttaact tggtttagct ttgccttgtt ctcagtcaca ttgataaaaa 94800tgtaagactt ttgtttatct tttagaataa tgacaccttt tggtgctgag aattttttgt 94860tttatatata tatatatata tacgtaatat aaatacaaaa tatatttaaa tatgtataat 94920atttctcata cactttatgt aactttgtgt tcctgtttct ctattatctt ggcatgtttt 94980cttcaaatgg cacttcttaa cctcctaagg ttaataaatt tctttgtaat ggacttttgt 95040tttctaattc ctcagcgtat gacaaatgaa ttatactttg tcaaattatt taggtaactt 95100tcagtttttg aagtcctggg atcataacat tcatcagtct ttaatttctg tcattaaggt 95160cattagctat aaatgaattt atgagtagat ttaaaaaata aaacatacaa tccttccctt 95220aacacacttt cccaccattt ggttcaactg ctagtgtaaa agcatgatga attttgagaa 95280gttatatttt accagttact ttatttttta ccagttattt aaaacagaca tgagccaaag 95340ccagaatact tgttaatgaa aatgaggtgt tttggaggaa aggaaggttg tgctgcagtt 95400tttacttgaa atctgttaca tttctttaca gaaatttcaa atctcttgtt tcctgttatg 95460atggtggcat tatatacctt taaaatgtga gctataggaa aatgaatgat ggttaatttt 95520ttaataaata tttagacttg tgtttttgaa attttttata acattgttat aggttttatc 95580ctctttctct tgtgaacatg tagtgatttg tattttgtga tctttgccgc atgctagaga 95640cttaagaata ctatagcaaa tatctgtctt ctttacattt aaaaattttt cgtgactact 95700ccctgttgat atctgtctta aaagttactt ttgatgtagt tcacaaatgt accagataat 95760tatttcatcg tttttaatgc ttaaagtttt tatttgtatt aggattttta gtatgatttt 95820aatgttaaag ttttgaagtt actctgccac tagaagtcta attttgggac ttactattca 95880tgaaatagga attgactttt atataagtaa taggacctta ttttgaaggt tcaaactgga 95940gaaaatctta cattgtttat atttttattt catttatttc agttgatttg cttgagatca 96000agattgcaga tacagaatcc atatttcgtg tatattgctg atattaatca ttaaaatcgt 96060ttttgacagt ttgacagtta aaggcatttc ctgtgaaata atactggtat gtatttaacc 96120atgcagatcc tcagtttgtg gtctgccagc taaaggtgaa gatatattcc tccaattcag 96180gacccacacg acgggaagac aagttcatgt actttgagtt ccctcagccg ttacctgtgt 96240gtggtgatat caaagtagag ttcttccaca aacagaacaa gatgctaaaa aaggtttgta 96300ctttactttc attgggagaa atatccaaaa taaggacaga ttaaaagcta tattttattt 96360tatgacatgt aaggaactat aatttgtttt ctattagatc tgcaggtgtt ttgcttactc 96420tggcattggt gagacattat aagggtaaat aatcctgttt gaaggaaaag gccttatggc 96480attgtaacat gagaggaatt tttcttaaca aggatggtta actgagaaga aattagcatg 96540ggaccaatat tttaaaaatt ttggtctata ggtagaaatg agatctgttc tgtggtctta 96600tgtagtgaca caaaccactt tttctccatt ttggcttatg tttctttttc tttccttttt 96660tttttttttc ctttttgtta gagacagggt cttgttctat tgcccaggct gagtagctaa 96720gactacaagc atgtgccacc acacccagct aatttttttt atttttattt ttgtagggac 96780agggtctcac tatgttgccc aggctggtct caaactcctg ggcacaagca gtcctcacgc 96840tttggcatcc caaagagttg gaattacagg tgtgcgccat catgcctggc cttaacgttt 96900cttaagactt gattattttc tatttagctt ctgtggattt actgattaat tttttaacta 96960ggagagaaat cagtatgaag aggaagtaat aaagaatgaa aacatggtat ttaaatgtgc 97020aggtttagaa agttaatgaa gtttgaattt gattgatctg tatttagaga aggcaacgtc 97080ttattatttt aaaaccaact atccgccctg tgcggtggct cacgcctgta attccagcac 97140tttgggaggc tgaggtgggc agatcagctg aggtcaggag ttcgagacca gcctggccaa 97200catggttaaa ccccatctct actaaaaata caaaaaaatt agccgggtgt ggtggcaggc 97260gcctgttttc ccagctactc aggaggcttg aggcaggata attgctgaac ccgagaggcg 97320gaggttgcag taagccaaga atgcaccatt gtactccagc ctgggcaaca agagtgaaac 97380tccatctcaa aaaaaaagaa aaaaaaaaca acaactatct tcatttaaaa tattaaatgt 97440gaatatttaa agtgagacta aggtgcaaca tttttagata gtaatgaaga aaaggactaa 97500ctttgtagtg ttgctgcctt gttaaacata ctagatagca tattgccaat ctttaaacat 97560tctcaatgat aggatttatt tactttttct gatttttagc ttttcttttg aaagaaaata 97620agaggaagtt tcatttactg caaaatttta aatgctgctt tgatgtatca gtagagatat 97680aattttcctt tatccagaat ccaagtagct ggaaaaaaaa atcaaaatat gctgaacttt 97740ttttttttta gccagaaacc catttcctat cgtctgtaca aataaaagtt aaatatatct 97800caataactta gaaaaattat tttttgataa tccaggaagt attagcaact gttttaaaat 97860taagataact agtaagtttt atttagcttt caaaaatagg catctacatc atcatctctg 97920cataccttta ggaatttcct aattcttatt tcccttcatc tgtactttaa cacatgcaaa 97980attgaaggtt agattaaata tttatgattt atttgtttat ccttgactac ataaatttcc 98040attttattga ttttccctgc cttatttaag aatatgctat gattaaaaca caaaaaattt 98100tagtataacc catatatata tagaattcac ctttttgtta tttaaatatt attggcttat 98160tttcttctaa gtaaaataca attactggct aaaataattg aaataagcaa aaaaaaaatt 98220ttaaagacct tgtatacaag attactttgc caggtactgt taaaagatgc aatgacattt 98280aagacgtaac atccttaagg atcttatttt ctgggggata aaaaacttta agataaatta 98340gaataaaaga tttaaatggc attttaaggt accaggtacc agataagatg tcacaaggct 98400gtatatcatt aattgccaaa tgatttatac aggccagatt tctttgttgg tcaatagagg 98460tttaaagtga tgaacttctg ttgtgttttt ttattaagaa ggtattatct tattagtaag 98520aagtgatttt ttttaagaac aagcatttta taacatcaaa agaaatcagt agtactcttt 98580cctaccccct catatttatt ctgaaagtat tcaagcatta tattgtcatg taagaaactg 98640gagcttctca tgtttgtatt gctgtagaag taaacatgta tttgccatgc gtcatcaggg 98700aagttgcact caccgtccaa gaacttttgt taaagtaaat cttggaatag gtagctcatt 98760tgaaatgtag aaaaaattaa atccatatct gaattttgtt tatatgtatg tacacgtaaa 98820ctaaaaacgt atttaaagct agtattagat gagaaaagag gtttttttac ttaaaatttt 98880aaggcaaaag tagtttatct tagatcttgt gagattgtat ttttggttta aaatttgaga 98940atttgagtga agaaaaatca tgtgaatgaa aatgcaacag ataactcaga ttgccttata 99000atagtctttg tgtttacctt tattcagaat atcaaatgat agtttatttt gttgactttt 99060tgcaaatgtt taacataggt gacagatttt cttttttaaa aaaataaaac atcattaatt 99120aaatatgtca tttcatttct ttttcttttc tttttttttt tttttaggac aaaatgtttc 99180acttttgggt aaatacattc ttcataccag gaccagagga aacctcagaa aaagtagaaa 99240atggaagtct atgtgatcaa gaaatcgata gcatttgcag tatagagcgt gcagataatg 99300acaaggaata tctagtactt actttaacaa aaaatgatct tgacaaagca aataaagaca 99360aagccaaccg atacttttct ccaaatttta aggtcagtta aattaaacat tttgtggggg 99420ttgttgactt gtatgtatgt gatgtgtgtt taattctagg agtacagctg atgaagaact 99480tgcttgacaa gtttttaact tatgtattat ttcgaagcag tgtttacgta gcagtaacat 99540gaaagtttct aataaaatac ccaatgtaca cagcgtcaaa aaagctgcat ttttcctttt 99600cctaattctt cgttgtttgc tgaaatctgg ggcaaaggtg cgggaggggg ctaaatgact 99660gggatatgaa gtaggaatgg gagaggaaag aaatagatgg gaactcagtc atttgggaat 99720gattcatatg gaatgttttt actgcttcca ctcctgtctg ccttccaatt tattctcaat 99780ccctcagagt gatcttaaaa atagacttga ttgtgtcact tctgtttaca ctttataagg 99840accttgtgtt ttttttttta ccatgaccta caaggcccag cataatttag cacagggcta 99900cctcctacat cagcactagt caccttctct ccttgtttct tgagattcag tcatactggt 99960ctttcttcag ttcttcaaaa tgctaagctt ctgcctcttc tagtctttcc agttattttc 100020cttctccctg taccttttca tctcagcctt ttcccctgac cttccatagc tatcttcata 100080tttccagcct tagcttcaat ctcatattct ctgaagtcct ttgattgtcc tcccgttatt 100140ctttttttaa aaatcctatt tccttatatt gtatcttaga attatttggt ttgtttcatt 100200tttgcctatg tgtgatatat gtatttctac ataggtatat atatctactt atagacaaga 100260attcttcaga ttaaaaaaat ctgatttgta aacattccca agtggttgtt taccattttt 100320ttcttccccc ttcctatttc ttattctacc tgattttccc ctgttcattc accacactcg 100380tttctttctc ttttttactc tctcttaatt tttcattcaa tttttataac atgtaataaa 100440tctaactgta gcgtctgagt attaagaata ttgctagtaa tacttcacct gtaatcccag 100500cactttggga ggctaaggca ggcggatcac ttgaggccca ggagtttaag accagccggc 100560caatatggcg aaaccctatc tgcactacaa atacaaaaat tagctgggca tggtgtcgca 100620cacctgtaat cccagctact tgggaggctg aggcacaaga attgcttgag cctgtgagat 100680ggaggttgca gtgagccgag atcacaccag tgcacgtgca cttcagcctg ggcaacagag 100740caagactctg tcttaaaaaa aaaaaaaaaa aaaaaatata tacacacaca cacacacaca 100800cacacacaca cacacactat tactaccaat atacatacat atatgtatgt atgtatgtat 100860gtatattggt agtaatagta atacttgggc ccctgcacgt tttaagtgaa aatagatcta 100920atattaaatg tctttagccc ttaaattttt tttaagtgtt cagaagtttc cctttaaaaa 100980aatttttaat atataataat tgtacatatt tatgggatac agagtgatat tttcatgtat 101040gcagtgtgtg atgatcaaat caggataatt agcatatgga tcacctcaaa catttgtcat 101100ttctttgtgt taggaacatt caaaattctg tcttctagct atttgaaaat atacagtaaa 101160ttattgttga ctagttacag ttctatagaa cactataatt tattcctcct gtgtgtaatt 101220ttttatcttt taaccaacat ctccctatcc tcccctccca ctccctttcc cggcctctaa 101280taaccacact cttatgagct caactttttt agcttccata tatgagtgag aacatacggt 101340atttatcttt ctgtacctga cttattttac ttaacatcat gtcctccggg ctagacattc 101400tctttagaat ccacaggttt cctttctttt ctctaaatct gcattttgct cagccattaa 101460cttttaaaat gtctttttcc ctttagtttt attgttttct attttaatat tgcaagatgt 101520tttatatttg tgattacaaa taaaaactcc attattagta aacaaataca atgtcatata 101580gtagtaagtg ctataaaaaa tagacaggat agaaagtaat cttggtttgt atgttttttg 101640ttttttagca aagatgatta gagaaggccc aaccaagcag ataacattta agcagaggcc 101700taaatcatat aagtgagtta tacaaatatc tgggaaaaga gttaagagta cagatgcaaa 101760agcccttaga caagagaatg agcttggtat atctgaagag tggataagtc attttgactg 101820aaacagagtg gacaagaaaa ccagtccaag tgtaaagaca ctagtgtgtg ttcagcatag 101880gaaggatgta atctgaattt tgtgtttaat attccctgtg ttcatgcttt caaaatacag 101940atgagtgagg aaagtaggga gaaggggtaa taaaggaagc tgagagatca gttaagaggt 102000acttgaatag tttagtaaag atgagagaag atgtttgctt cttgttgccc ctcactgctt 102060agaatagtgg cagtgaaggg taacaagaag ctgtcagatt aacttaaaga gtttactgat 102120gcagtggatg ttggttgtaa gagaagaatt gataatgact cttggataat aggggaggga 102180ggggctgtca atataatata atgaagaagg gatttgaagt catttctgat ttaaatctca 102240catccactac ctacttttaa tagatatgta gcctttaaca agttccctaa cctttctggg 102300ccttagctac ctccccttgg aaatggaaat acctaacatg taaggttgtt ttgacagtta 102360ttttcactag gcatgtaaag gcacttgact ctctgttata gaccactgta ttatgttaat 102420gtccctctcc ttcctccctt taggtaaagt ttttagggct aataaatccc aaatatcaat 102480gttgatcagt agtttgtgtt tgtgtagtgt tgtttatatc aaaaactaca ttgaagccgg 102540gcacagtggt tcacgcctaa aatcgcaaca ctttgggagg ccaaggtggg cctcccacct 102600tgaactaagg agtttgagac cagcctgggc aacatggtga aatcccatct ctacaaaaaa 102660tataaaagct agctgggtgt ggtggcatgc acctgtagtc ctagctactt gggaggctga 102720ggttgatcct gggagtttga gcctgcagtg agctgtgaag atgccactgc actctagtct 102780gggtgacaga gcaagaccct gtctcaaaaa cacacacaca cacacacaca cacacaaaga 102840aatacattga tttttcacat aggtagtaag agaaacattc tttttgaact cagctgtttg 102900tgaattgaat tttgtaattc aaatgctata ttatgtaaac tattgatgac tttcaatctg 102960catttatttt gtataattat ttagttaata tttgccactt atattcctta aaaaataaaa 103020ttgaggttgg gcgtggtggc tcacacttgt aatcccagca ctttgggagg ctgaggcagg 103080cagattgcct gagctcagga gtttgagatc agcctgggca acatcatgaa ccccatttct 103140actaaaatac aaaaaattat ctgggcatgg tggtgtacac ctgtagccct agctgtttgg 103200gaggctaagg cacgagaatt gcttgaaccc gggaggcaga ggttgcagtg agccaagatc 103260atgccactgc actccagctt ggcaacagag caagactctt gtctccagaa ataaaaataa 103320ataaattgta ttaacatcct gatagtttat ctgtttagta cctagcaaga aagaaaatgt 103380tgaacatctt aagaagaggg tcatttaaaa ggcctcttaa agatcatgtt tgttacagtg 103440cttaaaaatt aatatgttca tctgcaaaat ggaataaaaa atctgttaaa aatatatttc 103500actaaatagt ttaagatgag tcatatttgt gggttttcat tttaaatttt ctttctctag 103560gtgaagctgt acttcacaaa aacagtagag gagccgtcaa atccagaggc tagcagttca 103620acttctgtaa caccagatgt tagtgacaat gaacctgatc attatagata ttctgacacc 103680actgactctg atccagagaa tgaacctttt gatgaagatc agcatacaca aattacaaaa 103740gtctgaattt ttttttatca agagggataa aacaccatga aaataaactt gaataaactg 103800aaaatggacc tttttttttt taatggcaat aggacattgt gtcagattac cagttatagg 103860aacaattctc ttttcctgac caatcttgtt ttaccctata catccacagg gttttgacac 103920ttgttgtcca gttgaaaaaa ggttgtgtag ctgtgtcatg tatatacctt tttgtgtcaa 103980aaggacattt aaaattcaat taggattaat aaagatggca ctttcccgtt ttattccagt 104040tttataaaaa gtggagacag actgatgtgt atacgtagga attttttcct tttgtgttct 104100gtcaccaact gaagtggcta aagagctttg tgatatactg gttcacatcc tacccctttg 104160cacttgtggc aacagataag tttgcagttg gctaagagag gtttccgaag ggttttgcta 104220cattctaatg catgtattcg ggttagggga atggagggaa tgctcagaaa ggaaataatt 104280ttatgctgga ctctggacca tataccatct ccagctattt acacacacct ttctttagca 104340tgctacagtt attaatctgg acattcgagg aattggccgc tgtcactgct tgttgtttgc 104400gcattttttt ttaaagcata ttggtgctag aaaaggcagc taaaggaagt gaatctgtat 104460tggggtacag gaatgaacct tctgcaacat cttaagatcc acaaatgaag ggatataaaa 104520ataatgtcat aggtaagaaa cacagcaaca atgacttaac catataaatg tggaggctat 104580caacaaagaa tgggcttgaa acattataaa aattgacaat gatttattaa atatgttttc 104640tcaattgtaa cgacttctcc atctcctgtg taatcaaggc cagtgctaaa attcagatgc 104700tgttagtacc tacatcagtc aacaacttac acttatttta ctagttttca atcataatac 104760ctgctgtgga tgcttcatgt gctgcctgca agcttctttt ttctcattaa atataaaata 104820ttttgtaatg ctgcacagaa attttcaatt tgagattcta cagtaagcgt tttttttctt 104880tgaagattta tgatgcactt attcaatagc tgtcagccgt tccacccttt tgaccttaca 104940cattctatta caatgaattt tgcagttttg cacatttttt aaatgtcatt aactgttagg 105000gaattttact tgaatactga atacatataa tgtttatatt aaaaaggaca tttgtgttaa 105060aaaggaaatt agagttgcag taaactttca atgctgcaca caaaaaaaag acatttgatt 105120tttcagtaga aattgtccta catgtgcttt attgatttgc tattgaaaga atagggtttt 105180tttttttttt tttttttttt tttttaaatg tgcagtgttg aatcatttct tcatagtgct 105240cccccgagtt gggactaggg cttcaatttc acttcttaaa aaaaatcatc atatatttga 105300tatgcccaga ctgcatacga ttttaagcgg agtacaacta ctattgtaaa gctaatgtga 105360agatattatt aaaaaggttt ttttttccag aaatttggtg

tcttcaaatt ataccttcac 105420cttgacattt gaatatccag ccattttgtt tcttaatggt ataaaattcc attttcaata 105480acttattggt gctgaaattg ttcactagct gtggtctgac ctagttaatt tacaaataca 105540gattgaatag gacctactag agcagcattt atagagtttg atggcaaata gattaggcag 105600aacttcatct aaaatattct tagtaaataa tgttgacacg ttttccatac cttgtcagtt 105660tcattcaaca atttttaaat ttttaacaaa gctcttagga tttacacatt tatatttaaa 105720cattgatata tagagtattg attgattgct cataagttaa attggtaaag ttagagacaa 105780ctattctaac acctcaccat tgaaatttat atgccacctt gtctttcata aaagctgaaa 105840attgttacct aaaatgaaaa tcaacttcat gttttgaaga tagttataaa tattgttctt 105900tgttacaatt tcgggcaccg catattaaaa cgtaacttta ttgttccaat atgtaacatg 105960gagggccagg tcataaataa tgacattata atgggctttt gcactgttat tatttttcct 106020ttggaatgtg aaggtctgaa tgagggtttt gattttgaat gtttcaatgt ttttgagaag 106080ccttgcttac attttatggt gtagtcattg gaaatggaaa aatggcatta tatatattat 106140atatataaat atatattata catactctcc ttactttatt tcagttacca tccccataga 106200atttgacaag aattgctatg actgaaaggt tttcgagtcc taattaaaac tttatttatg 106260gcagtattca taattagcct gaaatgcatt ctgtaggtaa tctctgagtt tctggaatat 106320tttcttagac tttttggatg tgcagcagct tacatgtctg aagttacttg aaggcatcac 106380ttttaagaaa gcttacagtt gggccctgta ccatcccaag tcctttgtag ctcctcttga 106440acatgtttgc catactttta aaagggtagt tgaataaata gcatcaccat tctttgctgt 106500ggcacaggtt ataaacttaa gtggagttta ccggcagcat caaatgtttc agctttaaaa 106560aataaaagta gggtacaagt ttaatgttta gttctagaaa ttttgtgcaa tatgttcata 106620acgatggctg tggttgccac aaagtgcctc gtttaccttt aaatactgtt aatgtgtcat 106680gcatgcagat ggaaggggtg gaactgtgca ctaaagtggg ggctttaact gtagtatttg 106740gcagagttgc cttctacctg ccagttcaaa agttcaacct gttttcatat agaatatata 106800tactaaaaaa tttcagtctg ttaaacagcc ttactctgat tcagcctctt cagatactct 106860tgtgctgtgc agcagtggct ctgtgtgtaa atgctatgca ctgaggatac acaaaaatac 106920caatatgatg tgtacaggat aatgcctcat cccaatcaga tgtccatttg ttattgtgtt 106980tgttaacaac cctttatctc ttagtgttat aaactccact taaaactgat taaagtctca 107040ttcttgtcat tgtgtgggtg ttttattaaa tgagagttta taattcaaat tgcttaagtc 107100cattgaagtt ttaattaatg ggcagccaaa tgtgaataca aagttttcag tttttttttt 107160tcctgctgtc cttcaaagcc tactgtttaa aaaaaaaaaa aaaaaaaaac atggcctgag 107220agtagagtat ctgtctactc atgtttaatt aaggaaaaac acttattttt agggctttag 107280tcatcacttc ataaattgta taagcacatt aaatagcgtt ctagtcctga aaaagtccaa 107340gattcttaga aaattgtgca tatttttatt atgacagatg tttgaagata attccccaga 107400atggatttga tactttagat ttcaattttg tggcttttgt ctattattct gtactctgcc 107460atcagcatat ggaaagcttc atttactcat catgacttgt gccatataaa aattgatatt 107520tcggaatagt ctaaaggact ttttgtactt gaatttaatc atgttgtttc taatattctt 107580aaaagcttga agactaaagc atatcctttc aacaaagcat agtaaggtaa taagaaagtg 107640tagtttgtac aagtgttaaa aaaataaagt agacaatgtt acagtgggac ttattatttc 107700aagtttacat tttctccatg taatttttta aaaagtaaat gaaaaaatgt gcaataatgt 107760aaaatatgaa gtgtatgtgt acacacattt tatttttcgg tatcttgggt atacgtatgg 107820ttgaaaacta tactggagtc taaaagtatt ctaatttata agaagacatt ttggtgatgt 107880ttgaaaaata gaaatgtgct agttttgttt ttatatcatg tcctttgtac gttgtaatat 107940gagctggctt ggttcagtaa atgccatcac catttccatt gagaatttaa aactcaccag 108000tgtttaatat gcaggcttcc aaaggcttat gaaaaaaatc aagaccctta aatctagtta 108060atttgctgct aacatgaaac tctttggttc ttttattttt gccagataat tagacacaca 108120tctaaagctt agtcttaaat ggcttaagtg tagctattga ttagtgctgt tgctagttca 108180gaaagaaatg tttgtgaatg gaaacaagaa tattcagtcc aaactgttgt aaggacagta 108240cctgaaaacc aggaaacagg ataatggaaa aagtctttta aagatgaaat gttggagcca 108300actttcttat agaattaatt gtatgtggct atagaaagcc taatgattgt tgcttatttt 108360tgagagcata ttattctttt atgaccataa tcttgctgtt tttccatctt ccaaaagatc 108420ttccttctaa tatgtatatc agaatgtggg tagccagtca gacaaattca tattggttgg 108480tagctttaaa aagtttgtaa tgtgaagaca ggaaaggaca aaatagtttg ctttggtggt 108540agtactctgg ttgttaagct aggtattttg agactacttc cccatcacaa caacaataaa 108600ataatcactc ataatcctat cacctggaga catagccatc gttaatatgt tagtgactat 108660acaatcatgt tttcttctgt atatccatgt atattcttta aaaatgaaat ttatactgta 108720cctgatctca aagcttttta gcttagtata tctgtcatga atttgtagga tgttccattg 108780catcagaaaa cggacagtga tttgattact ttctaatgcc acagatgcag attacatgta 108840gttattgaga atcctttcga attcagtggc ttaatcatga atgtctaaat attgttgaca 108900ttaggatgat acatgtaaat taaagttaca tttgtttagc atagacaagc ttaacattgt 108960agatgtttct cttcaaaaat catcttaaac atttgcattt ggaattgtgt taaatagaat 109020gtgtgaaaca ctgtattagt aaacttcatc acctttctac ttccttatag tttgaacttt 109080tcagtttttg tagttcccaa acagttgctc aatttagagc aaattaattt aacacctgcc 109140aaaaaaaggc tgctgttggc ttatcagttg tctttaaatt caaatgctca tgtgactttt 109200atcacatcaa aaaatatttc attaatgatt cacctttagc tctgaaaatt accgcgttta 109260gtaattatag tgggcttata aaaacatgca actctttttg atagttattt gagaattttg 109320gtgaaaaata tttagctgag ggcagtatag aacttataaa ccaatatatt gatattttta 109380aaacattttt acatataagt aaactgccat ctttgagcat aactacattt aaaaataaag 109440ctgcatattt ttaaatcaag tgtttaacaa gaatttatat tttttatttt ttaaaattaa 109500aaataattta tatttcctct gttgcatgag gattctcatc tgtgcttata atggttagag 109560attttatttg tgtggaatga agtgaggctt gtagtcatgg ttctagtgtt tcagtttgcc 109620aagtctgttt actgcagtga aattcatcaa atgtttcagt gtggttttct gtagcctatc 109680atttactggc tattttttta tgtacacctt taggattttc tgcctactct atccagttgt 109740ccaaatgata tcctacattt tacaaatgcc ctttcagttt ctattttctt tttccattaa 109800attgccctca tgtcctaatg tgcagtttgt aagtgtgtgt gtgtgtgtct gtgtgtgtgt 109860gaatttgatt ttcaagagtg ctagacttcc aatttgagag attaaataat ttaattcagg 109920caaacatttt tcattggaat ttcacagttc attgtaatga aaatgttaat cctggatgac 109980ctttgacata cagtaatgaa tcttggatat taatgaattt gttagtagca tcttgatgtg 110040tgttttaatg agttattttc aaagttgtgc attaaaccaa agttggcata ctggaagtgt 110100ttatatcaag ttccatttgg ctactgatgg acaaaaaata gaaatgcctt cctatggaga 110160gtatttttcc tttaaaaaat taaaaaggtt aattattttg actattcggt tttaaacttt 110220ttattcaaca aataccaagt ccctgctgta tatatgggtt tggaatacat tagtgatcaa 110280aacacaccct ctatccctgc ccttgaagag tttacactcc agtagaggaa acagaattta 110340cattaacaat tataaattgt gatacatgct gtggaaagtg aggtgcatgt gataatggtt 110400atcaaacatt atgctcccca aaatttccat caccttgcag ttgaatgagg cctatgtaac 110460tagtttattc taatgtaata cagaaagtag tgtgacactc cactttctgt aaaggcaatg 110520aaaaaaattt accatccagt ttctctcttc ccctactgct gtgattaagg attttgcatg 110580ttctagatgg tgcagctttg tcagcctggg tcttgagtga tactggagca gaactccctc 110640aaattgttcc ttctgccacg ctaaacttgt tagatattag cataaataag aaataaacca 110700tttagactga tactttggga caatttttta tcacagtgta agttatatcc tgactaaaac 110760agtgctttgg aaagatcagg gaagggtttc ttgagctaca aaagggagtt aagaatgaag 110820caaaatgaaa tatggcatct ttaaatgaca tgatgcaata aactctaaat tgcaagttat 110880tcaatagtta ctgagcacca gatatgtgtc agacactgtt ctagatgctt gggatacaag 110940tgggtgaaca gcaggacctt gccttcatgg agcttacatt ctagcaaggg aaaatagtca 111000acatactata tgtaaggtaa tgcgtgctat agcagaaatg aaaggtaagg gagctcagga 111060ttgctgggtg gagagatttc agtgttgaat ggtgtagtcc agaacaggcc tcactgggaa 111120ggtaagattt gaagactgct gactaacagt ggcattggga ttttccttca ttctttcaac 111180cagcgagatg cactcagaac cctctataca attttattct cactttatac aactctgtac 111240attagttctg agtttttaag atgattgttg gcatatctag ggagaatttt taggaatacc 111300acccaaaata gtcgcaatgt tctaaaacca ttttgttttc taaattggtt tggcaaactc 111360tatacaaaca gtctgttcct tttcattctc caagttgagt ttttaaattt aaattatact 111420ctttgtgcat tttgacaata cttacctctt ttcagaagac attaaagttc tgttatggcc 111480ttaaaacatt tctgtaaagc tgaaaggaaa tatcatttat gatttattga tgggaaacag 111540ttacaaattt tgagttactt gacaaacaga agggatgaaa tttatggcct agtgttgtgt 111600tttcattctt aacccataag caaaaatctc tggaaaagta ccaatatcta gtaaaagaaa 111660tattttttca caaaaagtat tttctatttt cccctcacct cttacctcgt ctcacctcaa 111720agttcaagaa gcttaacttt gtgatcaaag gaaaatagta taattgaata tttgatacta 111780tagaaatttg caactgtata agggcattat taaccttttg ctagtgatca gtttcctatc 111840agggcagcca cataaaatta ggcatgacac tttaaaagaa aatgttgact cttaactttt 111900cctcctctaa tgcttaattt taaaatagat ttaagcaact ttaaagacat tttcctgagc 111960aactgcattt tgttttcatt ctgaatttaa tgagactctt t 112001220DNAArtificial sequenceSynthetic oligonucleotide 2ctgctagcct ctggatttga 20320DNAArtificial sequenceSynthetic oligonucleotide 3ccttccctga aggttcctcc 20420DNAArtificial SequenceSynthetic oligonucleotidemisc_feature(1)..(9)bases at these positions are RNAmisc_feature(11)..(11)bases at these positions are RNAmisc_feature(13)..(20)bases at these positions are RNA 4cugcuagcct ctggauuuga 20520RNAArtificial sequenceSynthetic oligonucleotide 5cugcuagccu cuggauuuga 20

Claims

1. A method of reducing the size or amount of protein aggregates in a cell comprising: contacting a cell with a compound comprising a modified oligonucleotide, thereby reducing the size or amount of protein aggregates in the cell.

2. A method of inhibiting the formation of protein aggregates in a cell comprising: contacting a cell with a compound comprising a modified oligonucleotide, thereby inhibiting the formation of protein aggregates in the cell.

3. A method of increasing the liquid miscibility of a protein in a cell comprising: contacting a cell with a compound comprising a modified oligonucleotide, thereby increasing the liquid miscibility of a protein in the cell.

4. The method of claim 3, wherein the protein is in a protein aggregate.

5. The method of claim 3, wherein the liquid miscibility of the protein aggregate in the cell is increased.

6. The method of claim 4 or 5, wherein the size or amount of protein aggregates in the cell is reduced.

7. A method of modulating the sub-cellular distribution of at least one protein in a cell comprising: contacting a cell with a compound comprising a modified oligonucleotide, thereby modulating the sub-cellular distribution of at least one protein in the cell.

8. The method of claim 7, wherein the modulation of sub-cellular distribution of the at least one protein is an increase in the ratio of nuclear to cytoplasmic distribution of the at least one protein.

9. The method of claim 7 or 8, wherein the at least one protein is in a protein aggregate.

10. The method of claim 9, wherein the size or amount of protein aggregates in the cell is reduced.

11. The method of any of claim 1, 2, 4-6, 9, or 10, wherein the protein aggregates are present in the cytoplasm of the cell.

12. The method of claim 11, wherein the protein aggregates comprise an RNA-binding protein.

13. The method of claim 12, wherein the RNA-binding protein is FUS, TDP-43, or PSF.

14. The method of claim 12 or 13, wherein the RNA-binding protein comprises a mutation.

15. The method of claim 14, wherein the mutation is a point mutation.

16. The method of claim 14, wherein the mutation is an expanded repeat.

17. The method of claim 14, wherein the mutation is a deletion.

18. The method of any of claims 14-17, wherein the mutation causes protein aggregation, liquid immiscibility, and/or mislocalization of the protein in a cell.

19. The method of any of claims 12-18, wherein the RNA-binding protein comprises a low complexity domain.

20. The method of claim 19, wherein the modified oligonucleotide binds to the low complexity domain.

21. The method of any of claims 12-20, wherein the RNA-binding protein comprises an RNA recognition motif.

22. The method of claim 21, wherein the modified oligonucleotide does not bind to the RNA recognition motif.

23. The method of claim 21, wherein the modified oligonucleotide binds to the low complexity domain with higher affinity than it binds to the RNA recognition motif.

24. The method of any of claims 1-23, wherein the cell comprises a protein comprising an expanded repeat.

25. The method of any of claims 1-24, wherein the cell comprises RAN translation products.

26. The method of any of claim 1, 2, 4-6, or 9-25, wherein the protein aggregate is a messenger ribonucleoprotein granule.

27. The method of claim 26, wherein the protein aggregate is a stress granule

28. The method of claim 26, wherein the protein aggregate is a processing body.

29. The method of any of claim 1, 2, 4-6, or 9-28, wherein the protein aggregate comprises G3BP protein.

30. The method of any of claims 1-29, wherein the modified oligonucleotide is a gapmer, wherein the gap consists of linked 2'-deoxynucleosides and the wings consist of linked nucleosides comprising modified sugar moieties.

31. The method of any of claims 1-30, wherein the modified oligonucleotide comprises at least one modified sugar moiety.

32. The method of claim 31, wherein the at least one modified sugar moiety is a cEt modified sugar moiety, a 2'-MOE modified sugar moiety, or a 2'-fluoro modified sugar moiety.

33. The method of claim 31, wherein the at least one modified sugar moiety is a 2'-fluoro modified sugar moiety.

34. The method of any of claims 1-33, wherein the modified oligonucleotide comprises at least one phosophorothioate internucleoside linkage.

35. The method of claim 34, wherein each internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage.

36. The method of any of claims 1-35, wherein the modified oligonucleotide comprises at least one modified nucleobase.

37. The method of claim 36, wherein the at least one modified nucleobase is a 5-methyl cytosine.

38. The method of any of claims 1-37, wherein the nucleobase sequence of the modified oligonucleotide is not 100% complementary to a pre-mRNA or a mRNA in the cell.

39. The method of any of claims 1-38, wherein the compound comprises a conjugate group.

40. The method of any of claims 1-39, wherein the protein or protein aggregate is not a prion protein or prion protein aggregate.

41. The method of any of claims 1-40, wherein the cell is in an animal.

42. The method of any of claims 1-40, wherein the cell is in a human patient.

43. The method of claim 42, wherein the patient has a neurodegenerative disease.

44. The method of claim 42, wherein the patient has ALS.

45. The method of claim 42, wherein the patient has Alzheimer's Disease.

46. The method of claim 42, wherein the patient has juvenile onset ALS.

47. The method of claim 42, wherein the patient has Parkinson's Disease.

48. The method of claim 42, wherein the patient has frontotemporal dementia.

49. The method of claim 42, wherein the patient has Pick's Disease.

50. The method of any of claims 42-49, wherein at least one symptom in the patient is ameliorated.

51. The method of any of claims 42-50, wherein the patient's disease is treated or ameliorated.

52. The method of any of claims 1-51, comprising contacting a cell with a second compound comprising a modified oligonucleotide, wherein the second modified oligonucleotide is 100% complementary to a target nucleic acid in the cell.

53. The method of claim 52, wherein the target nucleic acid is a pre-mRNA or a mRNA.

54. A modified oligonucleotide for use in treating or ameliorating a neurodegenerative disease in a human in need thereof, wherein the modified oligonucleotide causes a reduction in the size or amount of cytoplasmic protein aggregates in the human.

55. Use of a modified oligonucleotide capable of causing a reduction in the size or amount of cytoplasmic protein aggregates in a cell for treatment of a neurodegenerative disease.

56. The method of any of claim 1-37 or 39-51, wherein the nucleobase sequence of the modified oligonucleotide is less than 70% complementary to a pre-mRNA or a mRNA in the cell.